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doi: 10.1016/j.molimm.2019.07.003.

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China.

² State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China. Electronic address: vet@mail.hzau.edu.cn.

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doi: 10.1016/j.molimm.2019.07.003.

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China.

² State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China. Electronic address: vet@mail.hzau.edu.cn.

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus causing diarrhea and intestinal damage in nursing piglets. Previous work showed that PDCoV infection inhibits type I interferon (IFN) production. To further identify and characterize the PDCoV-encoded IFN antagonists will broaden our understanding of its pathogenesis. Nonstructural protein 15 (nsp15) encodes an endoribonuclease that is highly conserved among vertebrate nidoviruses (coronaviruses and arteriviruses) and plays a critical role in viral replication and transcription. Here, we found that PDCoV nsp15 significantly inhibits Sendai virus (SEV)-induced IFN-β production. PDCoV nsp15 disrupts the phosphorylation and nuclear translocation of NF-κB p65 subunit, but not antagonizes the activation of transcription factor IRF3. Interestingly, site-directed mutagenesis found that PDCoV nsp15 mutants (H129A, H234A, K269A) lacking endoribonuclease activity also suppress SEV-induced IFN-β production and NF-κB activation, suggesting that the endoribonuclease activity is not required for its ability to antagonize IFN-β production. Taken together, our results demonstrate that PDCoV nsp15 is an IFN antagonist and it inhibits interferon-β production via an endoribonuclease activity-independent mechanism.

Keywords: Endoribonuclease activity; Interferon production; Nonstructural protein 15 (nsp15); Porcine deltacoronavirus.

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viral nonstructural proteins / immunology*

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national center for biotechnology information

# data[].attributes.attachment_link.title
Porcine Deltacoronavirus nsp15 Antagonizes Interferon-β Production Independently of Its Endoribonuclease Activity - PubMed

# data[].attributes.attachment_link.description
Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus causing diarrhea and intestinal damage in nursing piglets. Previous work showed that PDCoV infection inhibits type I interferon (IFN) production. To further identify and characterize the PDCoV-encoded IFN antagonists will broaden our ...

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Abstract

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the virus that caused the global pandemic that was first reported [ 1] on December 31, 2019 [ 2]. Taxonomically, SARS CoV-2 belongs to the realm Riboviria, order Nidovirales, suborder Cornidovirineae, family Coronaviridae, subfamily Orthocoronavirinae, genus Betacoronavirus (lineage B), [ 3] subgenus Sarbecovirus, and the species Severe acute respiratory syndrome-related coronavirus.

The genome of SARS CoV-2 (NCBI Reference Sequence: NC_045512.2) [ 4] is similar to the genome of the coronavirus that caused the SARS epidemic in 2003 (SARS CoV, NCBI Reference sequence: NC_004718.3) [ 5, 6]. Much of the understanding of the proteins found in SARS CoV-2 are based on the numerous research studies reported on SARS CoV and other related viruses (e.g. MERS CoV) [ 7, 8]. However, among the recent coronavirus outbreaks in the new millennium (SARS CoV: 2002-2003, MERS CoV: 2012, SARS CoV-2: 2020), SARS CoV-2 mysteriously had the most devastating global impact. Understanding the proteins present in these viruses enable a more rational approach to designing more effective antiviral drugs [ 9, 10]. The majority of proteins of SARS CoV have been characterized in detail. The proteins of SARS CoV consist of two large polyproteins: ORF1a and ORF1ab (that proteolytically cleave to form 16 nonstructural proteins), four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and eight accessory proteins: ORF3a, ORF3b (NP_828853.1, not present in SARS CoV-2), ORF6, ORF7a, ORF7b, ORF8a, ORF8b, and ORF9b (NP_828859.1, not present in SARS CoV-2). Although accessory proteins have been viewed as dispensable for viral replication in vitro, some have been shown to play an important role in virus-host interactions in vivo [ 11]. Similar to SARS CoV, SARS CoV-2 lacks the hemagglutinin esterase gene, which is found in human coronavirus (hCoV) HKU1, a lineage A betacoronavirus [ 3]. The spike protein, envelope protein, membrane protein, nucleocapsid protein, 3CL protease, papain like protease, RNA polymerase, [ 10] and helicase protein have been suggested to be viable antiviral drug targets [ 12]. SARS CoV-2 is an RNA virus and its RNA genome is 30 kb in length. SARS CoV-2 is thought to have originated from its closest relative, BatCov RaTG13 (GenBank: MN996532), [ 13] which was isolated from horseshoe bats [ 14].

Discussion: Proteins of SARS CoV-2

SARS CoV-2 (NC_045512.2) has a total of 11 genes with 11 open reading frames (ORFs) (Table 1): ORF1ab, ORF2 (Spike protein), ORF3a, ORF4 (Envelope protein), ORF5 (Membrane protein), ORF6, ORF7a, ORF7b, ORF8, ORF9 (Nucleocapsid protein), and ORF10.

Polyprotein Expressed by ORF1ab

The first gene (ORF1ab) expresses a polyprotein. The ORF1ab polyprotein is comprised of 16 nonstructural proteins (NSPs) (Table 2).

NSP1 (Leader Protein)

Nonstructural protein 1 (NSP1) is the first protein of the polyprotein of SARS CoV-2 (Fig. 1-sequence alignment of NSP1 for SARS CoV with SARS CoV-2). This protein is also known as the leader protein. This protein is also found in SARS coronavirus and is known to be a potent inhibitor of host gene expression. NSP1 binds to the 40S ribosome of the host cell to inactivate translation and promotes host mRNA degradation selectively, while the viral SARS CoV mRNA remain intact [ 15]. Figure 1 shows the amino acid sequence alignment for the NSP1 proteins of SARS CoV (from genome: NCBI Reference Sequence: NC_004718.3) and SARS CoV-2.

Alignment of the primary amino acid sequence of NSP1 of SARS CoV (top, NP_828860.2) and SARS CoV-2 (YP_009725297.1). Sequence identity: 84.4%. Sequence similarity: 93.9%-determined using LALIGN software (and for subsequent alignments, Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26, see Supporting Information for output data) [ 16].

NSP2

Nonstructural protein 2 (NSP2) is the second protein of the polyprotein of SARS CoV-2 (Fig. 2). This protein is conserved in SARS CoV, the related beta coronavirus to SARS CoV-2. In SARS CoV, NSP2 was found to bind to two host proteins: prohibitin 1 and prohibitin 2 (PHB1 and PHB2) [ 17]. PHB1 and PHB2 proteins are known to play roles in cell cycle progression, cell migration, cellular differentiation, apoptosis, and mitochondrial biogenesis. The binding of NSP2 to PHB1 and PHB2 proteins suggest that NSP2 plays a role in disrupting the host cell environment.

NSP3 (Papain like Proteinase)

NSP4 (Contains Transmembrane Domain 2)

NSP4 interacts with NSP3 and possibly host proteins to confer a role related to membrane rearrangement in SARS CoV. Moreover, the interaction between NSP4 and NSP3 is essential for viral replication [ 18]. The sequence alignment for NSP4 proteins for SARS CoV and SARS CoV-2 is shown in Fig. 4.

NSP5 (3C-like proteinase)

The NSP5 protein based on the Middle East Respiratory Syndrome (MERS) coronavirus has been characterized. NSP5 cleaves at 11 distinct sites to yield mature and intermediate nonstructural proteins (NSPs) [ 21]. The amino acid sequence alignment for NSP5 of SARS CoV and SARS CoV-2 is shown in Fig. 5.

NSP6 (Putative Transmembrane Domain)

NSP7

NSP7 is required to form a complex with NSP8 (next section) and NSP12 to yield the RNA polymerase activity of NSP8 [ 24]. The primary amino acid sequence alignment for the NSP8 proteins for SARS CoV and SARS CoV-2 is shown in Fig. 7. Only one amino acid residue is different (arginine vs. lysine) but the charge is conserved at this location.

NSP8

NSP8 is a peptide cofactor that makes a heterodimer with NSP7 (the other peptide cofactor), and this NSP7-NSP8 heterodimer complexes with NSP12. In addition to the NSP7-NSP8 heterodimer, an NSP8 monomer unit also complexes with NSP12, which ultimately forms the RNA polymerase complex. The cryo-EM structure of this complex has been solved [ 25]. The amino acid sequence alignment for NSP8 of SARS CoV and SARS CoV-2 is shown in Fig. 8.

NSP9

NSP9 from the porcine reproductive and respiratory syndrome virus (PRRSV) has been found to interact with the DEAD-box RNA helicase 5 (DDX5) cellular protein [ 26]. This interaction between NSP9 and DDX5 has been shown to be important for viral replication-when the DDX5 gene was silenced in MARC-145 cells, the virus titers were lower by tenfold. Figure 9 shows the amino acid sequence alignment between the two NSP9 proteins from SARS CoV and SARS CoV-2.

NSP10

NSP10 has been shown to interact with NSP14 in SARS coronavirus, and this interaction stimulates activity of NSP14. NSP 14 is known to function as an S-adenosylmethionine (SAM)-dependent (guanine-N7) methyl transferase (N7-MTase) [ 27]. Furthermore, NSP10 has also been shown to stimulate the activity of NSP16, which is a 2′-O-methyltransferase [ 28]. Figure 10 shows the amino acid sequence alignment between the two NSP10 proteins from SARS CoV and SARS CoV-2.

NSP11

The function of NSP11 seems to be unknown. NSP11 is made of thirteen amino acids and the first nine amino acids (sadaqsfln) are identical to the first nine in NSP12. Figure 11 shows the amino acid sequence alignment between the two NSP12 proteins from SARS CoV and SARS CoV-2.

NSP12 (RNA Dependent RNA Polymerase)

NSP12 is the RNA-dependent RNA polymerase that copies viral RNA. As mentioned, NSP12 makes a complex with an NSP7-NSP8 heterodimer and an NSP8 monomer to confer processivity of NSP12. NSP12 exhibits poor processivity in RNA synthesis-that is the presence of NSP7 and NSP8 lowers the dissociation rate of NSP12 from RNA [ 29]. The amino acid sequence alignment between the two NSP12 proteins from SARS CoV and SARS CoV-2 is shown in Fig. 12.

NSP13 (Helicase)

NSP14 (3′ to 5′ Endonuclease, N7-Methyltransferase)

NSP14 from coronavirus is known to have 3′-5′ exoribonuclease activity and N7-methyltransferase activity [ 33]. The guanine-N7-methyltransferase activity is part of the process for introducing the 5′-cap of the virus, which involves multiple steps: [ 1] the gamma-phosphate of the 5′end of nascent mRNA is removed by the RNA triphosphatase (NSP13), [ 32], [ 2] a GMP moiety derived from a covalent enzyme-GMP intermediate is transferred to the resulting mRNA with a diphosphate end, [ 3] the GpppA cap is methylated with S-adenosyl-methionine, which is catalyzed by the guanine-N7-methyltransferase (NSP14) to yield the cap-0 structure, [ 34] and [ 4] 2′-O-methylation by NSP16 of adenine gives the cap-1 structure [ 35]. It is currently unknown which enzyme incorporates the GMP group involved in the second step, and it is possible that the virus uses the host guanylyltransferase enzyme [ 36]. Figure 14 shows the amino acid sequence alignment between the NSP14 proteins of SARS CoV and SARS CoV-2.

NSP15 (endoRNAse)

NSP15 of SARS coronavirus has been biochemically characterized as an endoribonuclease that cleaves RNA at uridylates at the 3′-position to form a 2′-3′ cyclic phosphodiester product [ 37]. The NSP15 protein specifically targets and degrades the viral polyuridine sequences to prevent the host immune sensing system from detecting the virus [ 38]. The crystal structure of NSP15 has been reported for SARS CoV [ 39] and SARS CoV-2 [ 40]. NSP15 uses manganese as a cofactor to promote endoribonuclease activity [ 41]. It has been suggested that NSP15 degrades viral dsRNA to prevent host recognition [ 42]. The amino acid sequence alignment of NSP15 from SARS CoV and SARS CoV-2 is shown in Fig. 15.

NSP16 (2′-O-Ribose-Methyltransferase)

Spike Protein (Surface Glycoprotein)

The spike protein (Fig. 17-sequence alignment between SARS CoV and SARS CoV-2) is a glycoprotein, which mediates attachment of the virus to the host cell. The structure of the spike (S) protein has been determined. This protein recognizes the human angiotensin-converting enzyme 2 (ACE2) protein on the host cell surface [ 45, 46, 47]. SARS CoV spike mouse polyclonal antibodies potently inhibited SARS CoV-2 spike protein mediated entry into cells [ 47]. Interestingly, a furin cleavage site (highlighted in Fig. 17: QTQTNSPRRARSVASQSIIA) was located in the S protein of SARS CoV-2, which was lacking in the S protein of SARS CoV. This difference in site could possibly explain the difference in pathogenicity of these two viruses [ 47].

ORF3a Protein

The ORF3a protein from SARS CoV is an ion channel protein related to NLRP3 inflammasome activation. ORF3a interacts with TRAF3, which in turn activates ASC ubiquitination, and as a result, leads to activation of caspase 1 and IL-1β maturation [ 48]. The amino acid sequence alignment between the two ORF3a proteins from SARS CoV and SARS CoV-2 is shown in Fig. 18.

Envelope Protein

The envelope protein is a small integral membrane protein in coronaviruses, which can oligomerize and create an ion channel [ 49]. The four structural proteins of coronaviruses are: S protein, M protein, E protein, and N protein [ 50]. The E protein has been shown to play multiple roles in the viral replication cycle: [ 1] viral assembly, [ 51] [ 2] virion release, [ 52] and [ 3] viral pathogenesis [ 53]. Interestingly, in the sequence alignment of the E proteins from SARS CoV and SARS CoV-2 (Fig. 19), there is a glutamate residue (E69) with a negative charge in SARS CoV that corresponds to a positively charged arginine in SARS CoV-2 (R69).

Membrane Protein

ORF6 Protein

ORF7a Protein

ORF7a from SARS coronavirus is an accessory protein that is a type I transmembrane protein and its crystal structure has been determined [ 59]. Figure 22 shows the amino acid sequence alignment between the two ORF7a proteins of SARS CoV and SARS CoV-2.

ORF7b Protein

The ORF7b accessory protein from SARS coronavirus is localized in the Golgi compartment [ 60]. Figure 23 shows the sequence alignment between the two ORF7b proteins of SARS CoV and SARS CoV-2.

ORF8 Protein

Nucleocapsid Protein

The nucleocapsid (N) protein of coronaviruses is a structural protein that binds directly to viral RNA and providing stability [ 63]. Furthermore, the N protein of SARS CoV-2 (Fig. 24) has been found to antagonize antiviral RNAi [ 64]. In another study, the nucleocapsid protein of SARS CoV was found to inhibit the activity of cyclin-cyclin-dependent kinase (cyclin-CDK) complex. Inactivation of the cyclin-CDK complex results in hypophosphorylation of the retinoblastoma protein and in turn inhibits S phase (genome replication) progression in the cell cycle [ 65]. Figure 25 shows the amino acid sequence alignment between the two N proteins of SARS CoV and SARS CoV-2.

ORF10 Protein

ORF10 protein from SARS CoV-2 is comprised of 38-amino acids and its function is unknown. Interestingly, SARS CoV possesses an ORF9b protein (NP_828859.1), which is not present in SARS CoV-2. Figure 26 shows the sequence alignment between ORF10 of SARS CoV-2 with ORF9b of SARS CoV. SARS CoV-2 does not have an ORF10 protein. A summary of the sequence identities and similarities of the discussed proteins from SARS CoV and SARS CoV-2 is shown in Table 4.

Overlapping Genes: ORF9b and Two Proteins with Variation Among SARS CoV-2 Sequences: ORF3b and ORF9c

Overlapping genes in coronavirus have been previously observed [ 67]. For example, in SARS CoV, the start and end positions in the nucleotide sequence of the N-protein are 28,120 and 29,388 respectively while the ORF9b gene of SARS CoV starts and ends at positions: 28,130 and 28,426 (within the gene sequence of the N-protein) [ 68]. Similarly, there is a putative ORF9b protein in SARS CoV-2 located within the gene encoding the N-protein, which does not yet have an accession number [ 4].

In the gene alignment of 2,784 SARS CoV-2 sequences, two variations were recognized in the SARS CoV-2 genome [ 66]. It was recognized that a premature stop codon at position 14 of ORF3b in SARS CoV-2 in 17.6% of isolates (position E14). Furthermore, there were two mutations that gave rise to premature stop codons in ORF9c (at position Q41 in 0.7% of sequences and at position Q44 in 1.4% of the sequences). The observations of these stop codons suggested that these genes for ORF3b and ORF9c may not be bonafide gene sequences in SARS CoV-2. With the putative SARS CoV-2 ORF3b protein, only 12 out of 57 overlapping amino acid residues were identical (21% sequence identity) to the ORF3b protein of SARS CoV [ 3]. In the above sections, ORF3b and ORF9c for SARS CoV-2 were not included in the above analysis. Another protein lacking an accession number is ORF14 [ 69].

Nontranslated (or Untranslated) Regions of SARS CoV-2 Genome

Considering the locations of each gene presented in Table 1, there are regions of the genome that are not translated into proteins, which is related to the non-canonical translational strategy employed by this virus [ 70]. The nucleotide sequences between the genes are the intergenic regions [ 71]. For instance, there is a conserved transctiption regulatory sequence (TRS) - a conserved hexanucleotide sequence: (5′-ACGAAC-3′) [ 71] that could be found in between some of the open reading frames (Table 5, Entries 2, 3, 4, 5, 7, and 9). This particular sequence has previously been identified as the leader-body fusion sites [ 71]. Furthermore, this sequence is a conserved motif that can be found in subgroup 2b, 2c, and 2d viruses [ 72]. Another transcriptional regulatory sequence was CUAAAC (e.g. Table 5, Entry 1) [ 73, 74].

Exploration of Treatment Options for COVID-19

An intense effort has been put forth to discover potential treatment options for COVID-19, the disease caused by SARS CoV-2 [ 75, 76, 77]. For instance, the FDA approved drug, ivermectin, is known to inhibit nuclear transport, and has been shown to inhibit the replication of SARS CoV-2 [ 78]. Other drugs have been repurposed and tested against COVID-19 [ 79, 80]. Remdesivir is a potential antiviral drug originally developed to treat ebola [ 81] and has been used to treat COVID-19 [ 82] by inhibiting viral RNA polymerase activity. Hydroxychloroquine [ 83] and chloroquine [ 84] have been used to potentially treat COVID-19. However, the use of these drugs has been known to result in cardiotoxicity [ 85, 86]. In fact, in a recent observational study, it was determined that hydroxychloroquine administration was not associated with a greatly lowered risk of death from COVID-19 [ 87].

A recent study identified 332 human proteins that interact with SARS CoV-2 proteins [ 66]. In this report, the predicted SARS CoV-2 proteins (NSPs 1-16 and ORFs) were expressed with 2xstreptavidin affinity tags. These tagged SARS CoV-2 proteins were expressed in human embryonic kidney (HEK)293T/17 cells and isolated the viral protein-(human protein) interactions using affinity purification-mass spectrometry. A total 332 protein-protein interactions (PPIs between SARS CoV-2 proteins and human proteins) were identified. Of these PPIs, 66 of them are targetable by compounds. Table 6 shows a set of compounds that target the identified PPIs based on chemoinformatics (entries 1-28) or expertise knowledge (entries 29-44). From the subset of potential antiviral compounds that were tested, two classes of compounds were found to be effective against viral pathogenesis: [ 1] protein translation inhibitors (i.e. zotatifin, ternatin-4, and PS3061), and [ 2] Sigma1 and Sigma2 receptor ligands (i.e. approved drugs: clemastine, cloperastine, and progesterone and PB28, which was ~ 20 times more potent than hydroxychloroquine with an IC ₉₀ of 280 nM in the viral titer assay is undergoing pre-clinical trials for anti-cancer [ 88] activity).

Table 6 Drugs that potentially target (modulate) proteins that interact with SARS CoV-2 proteins as described in reference [66]

Moreover, in another collaborative study, a library of 12,000 FDA-approved or clinical-stage drugs were tested against SARS CoV-2 infection in Vero-E6 (African green monkey kidney) cells [ 89]. Some effective compounds identified in the screen were: PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors (MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334), and MLN-3897 (a CCR1 antagonist).

Traditional Chinese Medicine (TCM) has also been employed in China to treat COVID-19 [ 90]. However, due to potential toxic components present in TCM remedies, [ 91, 92] the use of this strategy should be handled with caution [ 93]. Ironically, it has been suggested that TCM could have potentially been the cause of COVID-19 [ 94].

Conclusion

Although there is some variation in sequence in the proteins, many of the proteins found in SARS CoV-2 (NC_045512.2) are also found in SARS CoV (AY515512.1 or NC_004718.3) with 77.1% of the protein sequences shared in their proteomes [ 99]. Thus, previous research on related coronavirus proteins enable a better understanding of how we can approach to understand the current coronavirus (SARS CoV-2) that caused the current global pandemic (COVID-19). The general structures of most of the proteins from SARS CoV-2 can be visualized from homology models [ 100]. Advances in the knowledge of the structures and functions of the proteins in SARS CoV-2 will enable researchers to design better antiviral drugs that target this virus.

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Acknowledgements

Francis K. Yoshimoto, PhD holds a Voelcker Fund Young Investigator Award from the MAX AND MINNIE TOMERLIN VOELCKER FUND.

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# data[].attributes.attachment_link.title
The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19

# data[].attributes.attachment_link.description
The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.

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PAPER FABIO 1

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EndoU Activity Reduces the Accumulation of an Epitope Recognized by an Anti-dsRNA Antibody in CoV-Infected Hepatocytes.

Previously, we reported that EndoU activity delays the accumulation of an epitope recognized by the K1 antibody in the cytoplasm of IFNAR −/− bone marrow-derived macrophages (BMDMs) as measured by immunofluorescence ( 11). The K1 antibody was shown to recognize dsRNA; therefore, we hypothesized that the CoV epitope was dsRNA. To determine whether this phenotype is present in a stable cell line, we infected IFN-responsive AML12 hepatocytes with wild-type or EndoUmut MHV and measured accumulation of replication complexes (anti-nsp2/3) and dsRNA foci (anti-dsRNA, K1) at 8 h postinfection (hpi) ( Fig. 1A). In AML12 cells, wild-type and EndoUmut virus have similar replication kinetics and viral RNA expression, but EndoUmut elicited enhanced type I and type III IFN expression during infection (SI Appendix, Fig. S1). We quantified the number of nsp2/3 foci and dsRNA foci from 50 individual cells. We found that, while the numbers of nsp2/3-labeled replication complexes were not significantly different ( Fig. 1B, Right), the total number of dsRNA foci per cell was elevated in EndoUmut-infected cells ( Fig. 1B, Left). Median fluorescent intensity of the individual dsRNA foci was also brighter in EndoUmut-infected cells ( Fig. 1C). These results indicate that EndoUmut infection results in increased abundance of an epitope recognized by the K1 anti-dsRNA antibody.

The Viral RNA Recognized by the K1 Antibody during CoV Infection Is Negative-Sense RNA.

Since the RNA bound by the K1 antibody accumulates in the absence of EndoU activity, we sought to identify this RNA. To this end, we sequenced the RNA precipitated with the K1 anti-dsRNA antibody. We obtained ∼30 million reads for each total RNA sample and ∼10 million reads for immunoprecipitated samples. Upon mapping the reads to the mouse genome, we found similar read counts to host genes from both wild-type− and EndoUmut-infected cells (data available at NCBI GEO database, accession no. GSE144886) ( 33). We then mapped the reads to the MHV-A59 genome (GenBank accession no. AY910861) ( 34), and separated the viral reads by strand specificity, expecting to identify complementary sequences from positive- and negative-sense RNA. Surprisingly, we found that the majority of reads from the immunoprecipitated RNA sample mapped to negative-sense RNA ( Fig. 2A). We discovered that 99.8% of the reads from the input RNA sample mapped to positive-sense RNA. In contrast, 99.8% of the reads from the immunoprecipitated RNA mapped to negative-sense RNA. We found that the reads from the input RNA sample mapped across the entire MHV genome, as expected ( Fig. 2 B and D). Similarly, the reads from the immunoprecipitated RNA sample also mapped across the entire genome ( Fig. 2 C and E). We concluded that the K1 antibody immunoprecipitated full-length, negative-sense RNAs. When comparing the read counts between wild-type virus- and EndoUmut virus-infected samples, we found an eightfold increase (6 × 10 ⁵ read counts versus 4 × 10 ⁶ read counts) in the abundance of the reads from the EndoUmut virus-infected samples ( Fig. 2A). These results are consistent with the increase in dsRNA foci observed in EndoUmut-infected cells by immunofluorescence staining ( Fig. 1).

To determine the abundance of the dsRNA signal in other cell types, we infected IFNAR −/− BMDMs, C57BL/6 BMDMs, and AML12 cells with either wild-type or EndoUmut virus, and performed the anti-dsRNA immunoprecipitation experiment. We used random hexamers as primers for complementary DNA (cDNA) synthesis, which allows for generation of cDNA from both positive- and negative-sense RNA, and then evaluated the abundance of cDNA by qPCR. We consistently detected elevated levels of viral RNA immunoprecipitated by the dsRNA antibody from EndoUmut virus-infected cells as compared to the levels detected in wild-type virus-infected cells ( Fig. 3A). The total input viral RNA was similar between wild-type− and EndoUmut-infected cells ( Fig. 3B). Overall, our sequencing and qPCR results suggest that EndoU reduces the accumulation of a negative-sense viral RNA epitope that can be recognized by the anti-dsRNA antibody.

EndoU Activity Limits Abundance and Length of PUN RNAs.

Previous studies showed that the 5′ end of the CoV negative-sense RNA contains polyU extensions ( 35), and that EndoU cleaves at uridine residues ( 22, 25, 27⇓ ⇓ - 30). Therefore, we considered the PUN RNA as a potential target for EndoU activity. We hypothesized that PUN RNAs accumulate in the absence of EndoU activity. To quantitate the PUN RNAs, we generated cDNA from the negative-sense RNA using a strand-specific primer and performed a series of qPCRs with primers shown in Fig. 4A. Primer set 1 flanks a taqman probe and provides a measurement of total negative-sense RNA. Primer set 2 measures the PUN RNA. By normalizing set 2 to set 1, we can compare relative proportions of the negative-sense RNA that contain polyU sequences. To control for potential "self-priming" of the viral RNA during cDNA synthesis, we performed cDNA synthesis in the presence or absence of the negative-sense cDNA primer and quantified RNA expression by qPCR ( Fig. 4B). For both set 1 and set 2 qPCRs, we detected a significantly higher signal with the negative-sense primer compared to no primer. When comparing wild-type− and EndoUmut-infected cells, we detected a 10-fold increase in PUN RNAs from EndoUmut-infected cells as compared to wild-type virus-infected AML12 cells ( Fig. 4C, Left) and detected a 60-fold increase in IFNAR −/− BMDMs ( Fig. 4D, Left). To determine whether the polyA tail on the positive-sense RNA was similarly reduced by EndoU activity, we used either random hexamers or oligo-dT primers for reverse transcription and determined that the abundance of polyA tails on positive-sense RNA does not differ between wild-type and EndoUmut infections ( Fig. 4 C and D, Middle and Right). We concluded that EndoU activity reduces the abundance of negative-sense RNA that contains polyU extensions.

To determine whether EndoU reduces the lengths of the polyU extensions on the PUN RNA, we completed a nested PCR to obtain polyU-containing PCR products with a minimum predicted size of ∼100 base pairs (bp) ( Fig. 5A). We detected PCR species of ∼100 bp from both wild-type− and EndoUmut-infected cells, and detected a smear of larger PCR species unique to EndoUmut virus-infected cells ( Fig. 5B). To determine whether the length of polyA tails on the positive-sense RNA was affected by EndoU activity, we generated cDNA with oligo-dT primers to select for polyA-containing RNAs and performed the nested PCR reactions. We found that the products generated from positive-sense RNA were similar between wild-type and EndoUmut viruses, consistent with our previous results indicating that the polyA tail is not cleaved by EndoU activity ( Fig. 5C). To determine whether the smear of PCR amplicons represents extended polyU sequences, we sequenced the amplicons with next-generation sequencing and found that EndoUmut PCR amplicons had an increase in the number of reads and proportion of products with extended polyU sequences ( Fig. 5D). The most striking feature of the sequencing results is the bimodal distribution of the polyU extensions present in the EndoUmut-infected cell samples. We found that the majority (65%) of the reads from wild-type virus infection contained 10 uridine residues. In contrast, only 35% of the reads from the EndoUmut virus-infected sample contained 10 uridine residues. This was not due to a difference in the number of reads with 10 uridines but to an increase in longer polyU extensions detected in EndoUmut-infected cells. We detected variability in the polyU extensions in the EndoUmut virus-infected sample, with 65% of the reads containing from 11 to 17 uridine residues. We note that, while PUN RNAs in EndoUmut-infected cells are only a few uridines longer than the PUN RNAs from wild-type virus-infected cells, the PUN RNAs are 10-fold more abundant in EndoUmut virus-infected cells ( Fig. 4). Overall, these experiments revealed that EndoU activity reduces the abundance and length of polyU extensions on PUN RNAs, consistent with our hypothesis that EndoU cleaves the PUN RNAs during virus replication.

Since EndoU is conserved among CoVs, we sought to determine whether EndoU reduces the abundance and length of the PUN RNAs in the alpha-CoV porcine epidemic diarrhea virus (PEDV). Although the EndoU domains of MHV and PEDV exhibit only about 50% overall amino acid similarity, the catalytic histidines are 100% conserved ( 19). We showed that inactivation of EndoU in PEDV results in an increased type I and type III IFN response during infection ( 20). To determine whether EndoU limits the accumulation of PUN RNAs during PEDV infection, we infected cells with either wild-type or EndoUmut PEDV, isolated RNA, and evaluated the levels of PUN RNAs. We found that, relative to wild-type virus-infected cells, EndoUmut virus-infected cells contained abundant PUN RNAs in PK1 ( Fig. 6A) and Vero cells ( Fig. 6B). Sequences of PCR products templated by PUN RNA revealed that the length of the polyU extensions on the PUN RNAs was increased during EndoUmut virus infection ( Fig. 6 C and E), with a similar bimodal distribution of polyU extensions shown in Fig. 5D. During PEDV infection, we did not observe a difference in polyA tail length ( Fig. 6D). Taken together, these results indicate that PUN RNAs are generated during alpha- and beta-CoV replication, and that the highly conserved EndoU activity targets the polyU extensions in the PUN RNA.

PUN RNA Is a PAMP.

Since EndoU both reduces PUN RNA abundance and suppresses host MDA5 activation, we hypothesized that CoV PUN RNA is a PAMP. To directly test this hypothesis, we measured IFN stimulation following introduction of PUN RNAs derived from MHV-A59 into AML12 cells. PUN RNA was synthesized by T7 in vitro transcription of digested plasmids that contained sequences representing the 5′ end or 3′ end of the viral genome ( Fig. 7A). The PUN RNA, designated N5, and other CoV positive- and negative-sense RNA termini (P3, P5, N3) were transfected into AML12 cells. Total cellular RNA was harvested at 8 h posttransfection (hpt) and subjected to qPCR for IFNβ1 messenger RNA (mRNA) expression. We found that the presence of PUN RNAs increased IFNβ1 expression by 2,000-fold ( Fig. 7B), which was fourfold higher than any other in vitro transcribed viral RNA, indicating that PUN RNA is a PAMP.

To determine whether the polyU sequence contributed to the robust IFN stimulation of the PUN RNA, we transcribed PUN RNA containing either 12 uridines (N5) or no uridines (N5.NoU) at the 5′ end. We found that removing the 12 uridines from the PUN RNA significantly decreased the ability of that RNA to induce IFNβ1 expression ( Fig. 7C). Also, removing sections of the 3′ end of the PUN viral sequence (N5.180 and N5.100) resulted in a decrease in IFNβ1 expression, suggesting the polyU sequence alone is not sufficient to induce the IFN response ( Fig. 7C). Shortening the polyU extension to eight uridines (N5.8U) or four uridines (N5.4U) also diminished the IFN activation by the PUN RNA ( Fig. 7D). These results suggest that a polyU sequence of 12 uridines can enhance the IFN response to PUN RNA.

Previous studies documented that MHV-A59 infection induces IFN through MDA5 signaling ( 6, 11). To determine whether PUN RNA activates MDA5, we generated MDA5 knockdown (MDA5-KD) AML12 cells by CRISPR-Cas9 transduction ( Fig. 7E) and measured IFN activation by virus infection or RNA transfection. Both viral infection and the transfection of the PUN RNA induce IFNβ1 expression in an MDA5-dependent manner ( Fig. 7 F and G). During viral infection of MDA5-KD cells, both wild-type and EndoUmut virus infections had a significant reduction of IFNβ1 expression ( Fig. 7F). IFNβ1 induction by in vitro transcribed PUN RNA also was significantly reduced in MDA5-KD cells ( Fig. 7G). Importantly, we found that a single-stranded, in vitro-transcribed RNA activated MDA5, which was previously known to be activated by long complementary dsRNA. Taken together, these data suggest that the PUN RNA can act as an MDA5-dependent, viral PAMP.

EndoU Can Degrade PUN RNA and Dampen IFN Activation.

To determine whether EndoU activity can cleave the PUN RNA PAMP, we performed a series of in vitro cleavage assays ( 29). We incubated EndoU with 5′ negative-sense RNA containing a 12-uridine extension (RNA 1) or without the 12-uridine extension (RNA 2) ( Fig. 8A). When either RNA 1 or RNA 2 is mixed with EndoU in the presence of MnCl ₂, the RNA is degraded over time ( Fig. 8B). This degradation is most likely due to the presence of multiple uridines throughout RNA 1 and RNA 2, which is consistent with previous studies ( 22). We observed that EndoU cleaves RNA 1 more slowly than RNA 2 in this assay. We speculate that the polyU extension on RNA 1 may promote the formation of RNA secondary structures, which could contribute to the relative stability of RNA 1 versus RNA 2.

To determine whether the polyU extension can be cleaved, we substituted the viral sequence uridines with adenosines and generated RNA 3 and RNA 4 ( Fig. 8A). When mixed with EndoU and MnCl ₂, the polyU extension of RNA 3 is cleaved, producing a cleavage product the size of RNA 4 ( Fig. 8C). RNA 4 was not cleaved, consistent with the requirement of uridine residues for EndoU recognition and cleavage. To determine whether EndoU cleavage can decrease the ability of PUN RNA to stimulate IFN, we cleaved the PUN RNA with EndoU ( Fig. 8D). In the presence of EndoU and MnCl ₂, the PUN RNA was degraded into smaller RNA fragments. After EndoU treatment, we transfected the PUN RNAs into AML12 cells and measured IFN stimulation ( Fig. 8E). We found that transfecting the RNA treated with EndoU decreased the IFN stimulation activity. We note that the PUN RNA with MnCl ₂ migrated faster in the agarose gel, likely due to the addition of the Mn ²⁺ cation ( 36), but we do not observe a difference in IFN stimulation in the presence of MnCl ₂ alone. Overall, EndoU is capable of cleaving and degrading PUN RNA, which then reduces the ability of PUN RNA to stimulate IFN.

Cells, Viruses, and Reagents.

AML12 hepatocytes (CRL-2254; ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (12400-024, Invitrogen) supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (FBS), Insulin, Transferrin, and Selenium (41400045; Life Technologies), and Dexamethasone (40 ng/mL, D4902; Sigma). L929 cell line was gifted from Francis Alonzo, Loyola University of Chicago, Maywood, IL, and maintained in DMEM (10-017-CV; Corning) supplemented with 10% FBS, 1% l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin. Differentiated BMDMs were maintained in bone marrow macrophage media containing DMEM (10-017-CV; Corning) supplemented with 30% L929 cell supernatant, 20% FBS, 1% l-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin. Methods for generation of BMDMs are described in Deng et al. ( 11). Porcine kidney epithelial cells, PK1 (CL101; ATCC), were grown in growth medium containing modified Eagle medium (MEM) (10-010-CV; Corning) supplemented with 5% FBS, and 1% penicillin/streptomycin. Vero cells were grown in growth media containing MEM (41500-018; Gibco) supplemented with 10% FBS, 0.5% lactalbumin enzymatic hydrolysate (68458-87-7; Sigma), and 1% penicillin/streptomycin. Wild-type MHV strain A59 (GenBank accession no. AY910861) and EndoUmut (H262A) were previously generated by reverse genetics and full-genome sequenced ( 11). Infectious clones of wild-type PEDV or EndoUmut PEDV (H226A) were previously generated by reverse genetics and full-genome sequenced ( 20).

dsRNA Immunoprecipitation PCRs.

IFNAR −/− BMDMs, C57BL/6 BMDMs, or AML12 cells were infected with wild-type or EndoUmut virus at a multiplicity of infection (MOI) of 1. At indicated times postinfection, RNA was isolated with RNeasy kit (74104; Qiagen). One microgram of RNA was mixed with dsRNA antibody (K1; Scicons) or mouse anti-Beta actin (A00702-40; Genscript), RNA binding buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 20 U/mL Ribolock [EO0381; Thermofisher]). After overnight incubation, protein G beads (LSKMAGG02; Millipore) were added and incubated rotating for 4 h at 4 °C. Protein G beads were precipitated with magnets, then washed with cold binding buffer. RNA was purified off beads with RNeasy Kit and reverse-transcribed into cDNA with RT2 First Strand kit (330411; Qiagen). The qPCR was performed as described below.

For RNA-seq, RNA was processed by University of Chicago Genomics Facility. The cDNA sequencing libraries were generated with TruSeq Stranded Total RNA with Ribo-zero extraction (Illumina), then sequenced on Illumina HiSeq4000. RNA-seq reads were analyzed with Galaxy's online platform ( https://usegalaxy.org/). Reads were groomed, clipped, and mapped with Hisat2 to the wild-type MHV strain A59 (GenBank accession no. AY910861) or host genome (GRCm38 Ensembl build of the C57BL/6J). The number of reads at individual nucleotides was calculated by plotCoverage. RNA-seq data have been deposited and are available in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database (accession no. GSE144886).

qPCR.

RNA was isolated with RNeasy kit from cells at stated times postinfection. Isolated RNA was reverse-transcribed with RT2 First Strand Kit. The qPCR reactions were performed using RT2 Kit (330502; Qiagen). The qPCR reaction is 1) 95 °C for 10 min, 2) 95 °C for 15 s, 3) 60 °C for 1 min, and a repeat of steps 2 and 3 for 40 cycles.

PolyU Extension PCRs.

Cells were infected with wild-type or EndoUmut virus at an MOI of 1. At indicated times postinfection, RNA was isolated with RNeasy Kit. For strand-specific cDNA, 500 ng of RNA was reverse-transcribed with Omniscript (205113; Qiagen) with a reaction temperature of 50 °C to reduce self-primed cDNA synthesis ( 60). For negative-sense RNA, the cDNA primer was 5′-GAATTCTGGTGGTGCTGATGAAC-3′ for MHV and 5′-GCAGCATTGCTCTTTGGTG-3′ for PEDV. For total RNA, the cDNA primers were random hexamers, and positive-sense RNA primer was oligo-dT. The qPCR was performed with SsoAdvanced Universal Probes Supermix (1725281; Bio-Rad). The qPCR reaction was performed with an annealing temperature of 60 °C with either primer set PolyU qPCR set 1 or PolyU qPCR set 2. Primers are listed in SI Appendix, Table S1.

PolyU length nested PCRs were performed with PFU Ultra Polymerase (600380; Agilent) with an annealing temperature of 60 °C. The cDNA was generated as described above. For negative-sense RNA, the cDNA primer was 5′-GAATTCTGGTGGTGCTGATGAAC-3′ for MHV and 5′-GCAGCATTGCTCTTTGGTG-3′ for PEDV. For positive-sense RNA, the cDNA primer was 5′-GGGGATCCGCGGTTTTTTTTTT-3′. A PCR was performed with set 1 primers for PolyU length. Then the product was diluted 1:1,000 and utilized in a PCR with set 2 primers for PolyU length. PCR products were separated on a 10% polyacrylamide gel and stained with SYBR Green II dye (S7564; Thermofisher). Primers for cDNA synthesis and PCRs are listed in SI Appendix, Table S1. For polyU length sequencing, sequencing libraries were generated with custom amplicon primers with nextera XT indexes, and the amplicons were sequenced on an Illumina Miseq V2 500 with paired-end 250-bp reads. Reads were analyzed and mapped to viral genomes with Galaxy's online platform.

RNA Transfections.

The pCAGGs constructs were generated containing a T7 promoter, the viral genome segment, and a HindIII cleavage site. Viral sequences are listed in SI Appendix, Table S2. Plasmids were digested with HindIII-HF (R3104; NEB), then purified with Wizard Gel and PCR Purification Kit (A9282; Promega). RNA was in vitro-transcribed using a T7 RNA polymerase (EP0111; Thermofisher) and purified by LiCl precipitation. Five picomoles of RNA or Poly I:C (P1530; Sigma) was transfected into AML12 cells using Lipofectamine 2000 (11668027; Thermofisher). At 8 hpt, RNA was isolated using RNeasy kit and qPCR for IFNβ1 (PPM03594C; Qiagen), and 18s ribosomal RNA (rRNA) (PPM57735E; Qiagen) was performed as described above.

Generation of MDA5-Knockdown Cells.

A modified CRISPR/Cas9 protocol, based on the GeCKO system ( 61), was used to knock down the function of MDA5 in AML12 cells. Single-guide RNA was identified with Benchling (Benchling, Inc.) to target the Ifih1 gene. Sequence used for targeting Ifih1 was 5′-ATGGACGCAGATGTTCGTGG-3′. The cDNA versions of guide RNA were annealed and inserted into a pLentiCRISPRv2-puro (Addgene 52961) cassette between flanking BsmBI sites. Transducing particles (TPs) were generated by transfecting HEK-293T/17 cells with pLentiCRISPRv2-puro, pPax2, and pHEF-VSV-G and collecting supernatant. TPs were centrifuged at 1,000 × g for 10 min at 4 °C then filtered through a 0.45-μM filter (Millipore Sigma). AML12 cells were transduced with TPs, then incubated for 24 h at 37 °C in 5% CO ₂. Transduced AML12 cells were then selected with 1 μg/μL puromycin (InvivoGen) for 96 h. Puromycin-selected cells were then grown and cloned into a monoclonal population. Knockdown of MDA5 was determined by Western blot using rabbit anti-MDA5 (SAB3500356; Sigma) and mouse anti-actin (A00702-40; Genscript).

RNA Cleavage Assay.

Cleavage of RNA substrates was performed according to Kang et al. ( 29). Purified, wild-type EndoU was kindly gifted by C. Kao, formerly of Indiana University, Bloomington, IN, currently at Aligos Therapeutics, San Francisco, CA. Briefly, 1 μM RNA was mixed with EndoU in Cleavage Buffer (50 mM Tris pH7.5, 50 mM KCl, 1 mM dithiothreitol) with or without 5 mM MnCl ₂. Reactions were incubated at 30 °C for indicated time, and reactions were stopped by addition of RNA Gel Loading Buffer (B0363S; NEB) and incubation at 95 °C for 5 min. Reaction products were immediately loaded into a 10% polyacrylamide gel with Tris-Borate-ethylenediaminetetraacetic acid (EDTA) buffer or a 1% agarose gel with Tris-Acetate-EDTA buffer, and bands were separated by electrophoresis. Gels were then stained with SYBR Green II dye and visualized with a ChemiDoc XRS+ imager (Bio-Rad), and processed with Image Lab software (Bio-Rad).

Data Availability Statement.

RNA-seq data have been deposited and are available in the NCBI GEO database (accession number: GSE144886).

View Abstract

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Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors

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Cells carry sensors that are primed to detect invading viruses. To avoid being recognized, coronaviruses express factors that interfere with host immune sensing pathways. Previous studies revealed that a coronavirus endoribonuclease (EndoU) delays activation of the host sensor system, but the mechanism was not known.

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viral nonstructural proteins / metabolism*

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national center for biotechnology information

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Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction With the Structural Proteins - PubMed

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Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed infor ...

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Abstract

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a 'double-hit' whereby two CoV-unique protein elements of the replicase-transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the 'double-hit hypothesis'.

Keywords: : combination antiviral therapy, COVID-19, double-hit hypothesis, nsp15/NendoU inhibition, RNA substrate mimetics

Graphical abstract

Replicase-transcriptase complex of SARS-CoV-2 & 'double-hit hypothesis' (Key: CoV-coronavirus, PLpro-Papain-like protease, 3CLpro-3CL protease, RdRp-RNA-dependent RNA polymerase, NendoU-endoribonuclease, ExoN exoribonuclease, 2′-O-Mtase-2′-O-ribose methyltransferase)

As of early May 2020, there is no specific anti-COVID-19 therapy available (clinical management of severe acute respiratory infection when COVID-19 disease is suspected; WHO interim guidance version 1.2). We, among others, have posited that a combination strategy with 'repurposed drugs' could offer the best chance of combating the current COVID-19 pandemic gripping the world [ 1]. In other words, 'multiple drugs versus one virus' where different nodes of the virus-host interactome are simultaneously targeted with antivirals to achieve a synergistic effect [ 2]. Indeed, the case for combination antiviral therapy is becoming increasingly clear, as demonstrated by the modest clinical efficacy hitherto reported of monotherapy, including for the leading RdRp inhibitor remdesivir [ 3].

Such a strategy is not without historical precedence in modern medicine, even before the era of 'personalized medicine', the latter best exemplified by precision oncology. For example, HIV causing AIDS rapidly emerged into the global scene in a very similar pandemic-like fashion during the late 1980s and 1990s. The ensuing decade saw the utilization of this combination strategy with highly active antiretroviral therapy against the new HIV virus. The success of this is evident and AIDS is now considered a manageable, chronic condition if addressed early on in its natural history. As the scientific community scrambles to find a therapeutic solution to face the latest global viral threat in the form of SARS-CoV-2, it is prudent to remember this history's lesson and attempt to replicate it. As such, what could be novel approaches to realize combination therapy in the context of COVID-19?

Rationale for the double-hit hypothesis

To appreciate the multiple drugs versus one virus approach, we must first understand the dynamics of the 'host-virus protein-protein interactome'. Using integrative meta-analysis, Bösl et al. have demonstrated a glimpse into viral evasion mechanisms that target central nodes of the host-virus interactome [ 2]. Here, a seemingly 'hub-and-spoke model' of the host-virus protein-protein interactome emerges in which viruses across families and strains ubiquitously target core host proteins involved in multifunctional cellular pathways for maximal effect. This is of particular importance to smaller RNA viruses (as compared with DNA viruses) with more reliance on host cellular machinery to partly compensate for their higher mutation rate per strand copying [ 4]. RNA viruses such as SARS-CoV-2 rely on RNA-dependent RNA polymerases (RdRps) for faithful transmission of their genomes from host to host [ 5]. However, RdRps are known for their low fidelity of replication, resulting in an elevated rate of spontaneous mutations in RNA viruses [ 6]. Consequently, nucleoside analogs have become a backbone of modern antiviral therapy, exploiting base misincorporation into RNA viral genomes for their therapeutic effect.

Ribavirin is a nucleoside analog broad-spectrum antiviral agent (BSAA) with activity against a number of RNA and DNA viruses [ 7]. It is one of the longest standing antiviral agents included in the WHO's Model List of Essential Medicines (21st list, 2019). These rather promiscuous antiviral properties of ribavirin have been explained by at least five mechanisms; indirect mechanisms, including inosine monophosphate dehydrogenase inhibition affecting the cytoplasmic nucleotide pool and immunomodulatory properties, and direct mechanisms, including RNA capping interference, canonical RdRp inhibition and lethal RNA viral mutagenesis causing 'error catastrophe' [ 8, 9]. However, ribavirin has not demonstrated a significant clinical benefit during previous outbreaks of coronaviridae, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), upon meta-analysis [ 10-12]. This is corroborated by ribavirin's IC ₅₀ in the high micromolar range against human CoVs in vitro, which currently limits its use in this setting due to toxicity at pharmacologically potent doses [ 13, 14]. Notably, this is in contrast to ribavirin's efficacy particularly in combination therapy against a wide variety of viral infections including hepatitis C virus, respiratory syncytial virus and Lassa fever [ 15-17].

Why did a model BSAA with a repertoire of antiviral mechanisms against a number of RNA viruses fail in the face of previous human coronavirus respiratory syndromes? Briefly, work of Ferron et al. using SARS-CoV-1 offers an explanation how a coronavirus can overcome lethal mutagenesis with an endowed exonuclease (ExoN) activity by RNA nucleotide mismatch correction [ 18]. Albeit in vitro evidence, such observations may explain the proven efficacy of a broad-spectrum nucleoside analog against hepatitis C virus without any exonuclease activity and lack of efficacy against SARS or MERS in possession of exonucleases. However, it is perhaps important that we reassess to explore any other overlooked strategies that could potentiate the efficacy of a BSAA in general.

Extrapolating from SARS-CoV-1 & MERS-CoV

Rapid sequencing efforts have established that SARS-CoV-2 is approximately 80% identical to SARS-CoV-1 at the whole genome level [ 19, 20]. This similarity apparently extends to the pathophysiological level of viral replication cycle to some degree, where SARS-CoV-2 shares the same angiotensin-converting enzyme 2 cellular receptor for entry and primes its spike (S) protein with the transmembrane protease serine 2 [ 19, 21-23]. The viral genome can be broadly segmented into regions consisting of nonstructural, structural and accessory genes. Importantly, homology modeling of four key viral proteins (papain-like protease, 3CL protease, helicase and RdRps) suggest that active sites of these viral enzymes to be highly conserved across the three known human coronaviruses (CoVs) with pandemic potential [ 24, 25]. As such, it is a reasonable inference to extrapolate the pharmacology of antiviral agents previously tested against SARS and MERS to rationalize therapeutic options for COVID-19.

Out of the three viral gene regions, those encoding the 16 nonstructural proteins (nsps) perhaps reign supreme in the hierarchy of critical importance to the viral life cycle, as they function in the interphase between cellular entry and exit facilitating ongoing transmission of the virus. Many of these nsps assemble into a supercomplex known as the replicase-transcriptase complex (RTC) with multienzymatic properties that is critical for replication and transcription of subgenomic RNAs [ 26]. Even more unique to CoVs are the nsp15-NendoU and nsp14-ExoN ribonucleases (the former in particular), which can be considered as genetic markers of the Nidovirales order, distinguishing it from all other RNA viruses [ 18, 27]. The work of Ivanov et al. in 2004 following the SARS outbreak perhaps best exemplified the indispensability of nsp15 to a human CoV. A single-nucleotide mutation of nsp15 abolished its endonucleolytic activity and viral RNA synthesis. This observation has been corroborated further in vivo where CoVs expressing mutant nsp15 forms resulted in early robust induction of interferon, apoptosis of macrophages and stimulated a protective immune response leading to significantly attenuated disease in murine models [ 28]. Therefore, this endonuclease can be considered an Achilles' heel-type weaknesses and specific antiviral target [ 29]. However, other studies have indicated that the endoribonuclease activity of CoV nsp15 can be dispensable for the viral replication cycle, albeit required for optimal infection [ 30, 31]. In fact, the key contribution of this evolutionarily conserved endoribonuclease may be the efficient evasion of the innate immune response at the very site of RNA synthesis during the early phase of infection [ 28, 32]. By functioning as an interferon antagonist and evader of double-stranded RNA sensors in macrophages, nsp15 likely represents a primary strategy employed by a CoV to evade the intrinsic antiviral responses and gain a foothold in the host [ 33].

The S protein on the contrary, while crucial for initial viral entry into target cells, has been observed in some CoVs to directly transit to the cell surface without being assembled into virions prior to exocytosis [ 34]. There it mediates fusion between infected and uninfected cells leading to the formation of giant, multinucleated cells facilitating almost a 'paracrine mode of viral spread' in a target tissue. If this is a mechanism faithfully employed by SARS-CoV-2, there could be significant ramification for vaccine development, which represents a major focus of current global efforts. The S protein has intuitively received most attention as the best target for development of monoclonal antibodies. However, the aforementioned mechanism of viral spread could become implicated in circumventing detection and neutralization by antibodies.

Case for inhibiting nsp15/NendoU

The endoribonuclease activity of nsp15/NendoU involves uridylate-specific cleavage of RNAs [ 29, 35-37]. Function of the CoV endonuclease in relation to its viral life cycle has remained somewhat elusive. Evidence exists in animal models for an immunomodulating property during early viral infection in addition to its canonical endoribonuclease activity. Nsp 15 (among other nsps) seems to play a dominant role in suppressing the type I IFN (IFN-α/β)-associated innate immune response by infecting macrophages, thus avoiding detection of viral mRNA by double-stranded RNA sensors [ 28, 32, 38]. Evidence from SARS animal models indicates that it is a delayed yet exuberant IFN-I signaling with an accumulation of neutrophils, monocytes and macrophages in lungs that is the major precipitant of lung immunopathology [ 39]. It is suggested that this temporally dysregulated and late-enhanced innate immune response with an influx of myeloid cells preceding a 'cytokine storm of proinflammatory mediators' drives acute respiratory distress syndrome, which is the dominant cause of COVID-19 mortality [ 40]. Site-directed mutagenesis of the homologous NendoU domain in equine arterivirus (type of nidovirus) has demonstrated up to five log reduction in viral replication and subgenomic RNA accumulation [ 41]. Being an endonuclease and a potential 'suicide enzyme' for an RNA virus, NendoU activity thus is evidently under tight control during replication [ 42]. In toto, nsp15 can be considered a highly specific and unique target for anti-CoV therapeutics.

RNA mimetics as decoy inhibitory ligands against nsp15/NendoU

A high-resolution crystal structure by x-ray diffraction for the nsp15/NendoU of SARS-CoV-2 has now been preliminarily reported leading to some crucial observations [ 43]. In their Protein Data Bank entry of SARS-CoV-2 nsp15/NendoU (PDB ID: 6VWW), Kim et al. report 88% sequence identity and 95% similarity with its closest known homolog in SARS-CoV-1. MERS-CoV nsp15/NendoU, on the contrary, is somewhat more distant with only 50% sequence identity and approximately 65% similarity. More importantly, six key residues forming the catalytic site (His235, His250, Lys290, Thr341, Tyr343 and Ser294) are universally conserved among SARS-CoV-2, SARS-CoV-1 and MERS-CoV, both sequentially and conformationally. This suggests that all three homologs should have extremely similar if not identical substrate affinity. The functionally active form of nsp15 is a double-ring hexamer made of a dimer of trimers, which is consistent with structures of SARS-CoV-1 and MERS-CoV nsp15s [ 43]. Up to six preferentially single-stranded RNAs can engage the hexamer from top and bottom for cleavage (). Therefore, inhibitors of SARS-CoV-1 nsp15 can be expected to inhibit its homolog in SARS-CoV-2. It must be noted that the work of Ivanov et al. [ 29], cited here for primarily supporting nsp15 as a unique nidoviral genetic marker, employed a set of mutant derivatives of HCoV-229E NendoU with Ala substitutions of conserved residues across XendoU/NendoU family. HCoV-229E is a related human CoV which is known to cause common colds and falls in the Alphacoronavirus genus; this contrasts with the Betacoronavirus genus of SARS-CoV-2. As the new nsp15/NendoU crystallographic data from Kim et al. demonstrates, high sequence identity or similarity cannot be expected between HCoV-229E and SARS-CoV-2 given the evolutionary distance [ 43]. Therefore, direct comparison of the active site residues from these studies is difficult and thus necessitates site-directed mutagenesis studies with SARS-CoV-2.

This brings us back to previous work by Bhardwaj et al. in 2006 [ 36]. Using SARS-CoV in vitro models, it was demonstrated that nsp15 specifically cleaves 3′ of uridylate in 16-nt, favorably modified RNA sequences. In a series of elegantly designed experiments, derivatives of the 16-nt prototype RNA substrate (named R16.4) that cannot be efficiently cleaved by nsp15 could still act as competitive inhibitors of this enzyme. These derivatives (e.g., PT13, mR16.4) obtained by minor modifications to the prototype R16.4 sequence (5′GAAGCGAAACCCUAAG3′) retain the ability to interact with nsp15, thus acting as competitive inhibitors of NendoU activity. Single-stranded RNA oligonucleotides with multiple, unpaired uridylate bases had preferentially higher affinity binding to nsp15.

This raises the possibility of a novel approach where RNA substrate mimetics of nsp15 can be deployed as endonuclease inhibitors. An important question is whether combining such endonuclease inhibitors with nucleoside analogs (as backbone antiviral agents) would prove to have an additive or synergistic effect against CoVs. A further possibility is a 'triple-therapy regimen' combining two approved nucleoside analogs, for example ribavirin and sofosbuvir, a combination shown to have tight RdRp inhibition in a recent molecular docking study [ 44].

Future perspective

The leading edge of current global efforts to curb the COVID-19 pandemic is understandably the development of a vaccine. The WHO reported in April 2020, that over 70 candidate vaccines are at different stages of the translational process at an unprecedented speed. Indeed, a proof of principle has been demonstrated in a rhesus macaque model where recovery from infection with SARS-CoV-2 conferred immunity to rechallenge with the same strain [ 45, 46]. However, recent phylogenetic network analyses on reported SARS-CoV-2 genomic samples from around the world have found at least three central variants (A, B, C) indicating in human clonal evolution with possibly different lethality [ 45, 47, 48]. First, most present vaccine candidates have a specific target epitope of SARS-CoV-2, and such rapid antigenic drift of the virus could seriously jeopardize effective vaccination. Second, even an effective preventive vaccine could only perhaps reduce the global infection rate to a basal level, meaning likely cyclical re-emergence of outbreaks similar to seasonal influenza in the near future. This poses a provocative question: is a situation in which vaccine composition has to be constantly changed to suit the dominant virulent strains practically feasible or desirable? All in all, this further highlights the critical role of designing novel therapeutic strategies with antivirals.

Here, we have attempted to elaborate on the previous proposal for using a BSAA combination therapy for COVID-19. Our focus was a concept for further investigation, in other words utilizing already licensed BSAAs with some activity against SARS-CoV-2 concurrently with experimental RNA substrate mimetics as nsp15/NendoU inhibitors. To our knowledge, only in vitro evidence exists for such nuclease inhibitors to-date [ 36]. More lead optimization work on such RNA substrate mimetics to further establish proof of principle is now warranted at a minimum. These can include parallel cellular assays to characterize their stability within microsomes or nanoparticles and off-target properties. As such, the 'double-hit effect' is hypothesis-generating at present and additional supporting data would be necessary prior to advancement of this strategy into in vivo animal model studies and subsequent first-in-human clinical trials. Conversely, the CoV nsp14 would be an attractive target to combine with a BSAA; inhibition of the ExoN catalytic site can be expected to knock out this viral RNA proofreader, thus restoring the potency particularly of nucleoside analogs that are readily available antiviral agents [ 1, 18, 42, 47]. Unlike for NendoU, we are not yet aware of any reported inhibitor class specifically of the CoV exonuclease activity, which limits advancing this combination strategy for now.

We can only speculate on the challenges of RNA substrate mimetics vis-à-vis their translational potential. While the PK/PD profiles of long-standing nucleoside analogs are well established, the in vivo dynamics of potential RNA substrate mimetics are essentially unknown. Additionally, the optimal method or vehicle for their delivery remains to be determined. One possibility is the emerging RNA-based delivery system which can offer significant advantages in terms of simpler design and flexibility of manipulation in comparison to the more traditional viral subunit/vector-driven methods [ 49]. It is also noteworthy that mRNA is under investigation as a robust platform for at least one of the current leading vaccine candidates (mRNA-1273 by Moderna, Inc., MA, USA) which has rapidly been translated into Phase I clinical trial (ClinicalTrials.gov Identifier: NCT04283461). This involves a novel lipid nanoparticle-encapsulated mRNA formulation that encodes for the full-length S protein of SARS-CoV-2 which can be readily administered intramuscularly. In principle, given the relative ease of manufacturing RNA oligonucleotides compared with large recombinant viral proteins, we can anticipate quick adaptability of a similar delivery system to RNA substrate mimetics as cargo. Another important consideration is the temporality of administration of such antiviral therapy. As has been empirically noted, an initially dampened host innate immune response preceding a late exuberant cytokine storm are the hallmarks of severe COVID-19 immunopathology and associated mortality [ 50]. Given the current understanding of CoV-nsp15/NendoU as a major immunomodulator, counteracting viral evasion of host innate immunity in the preliminary stages of disease seems logical. Additionally, formation of double membrane vesicles and RTC activity dominate early during the coronavirus replication cycle before viral protein synthesis escalates to sufficient levels causing viremia and systemic infection. Therefore, the ideal time for inhibition of SARS-CoV-2 endonuclease activity within a combination strategy is perhaps quite early on in COVID-19.

Further, even if favorable PK/PD data are obtained through preclinical testing, the toxicity profiles of such therapeutics solo and in combination for drug-drug interactions would need to be rigorously evaluated in model in vivo systems. The 'double-hit hypothesis' may also dictate that SARS-CoV-2 proteases (i.e., PLpro and 3CLpro) could serve as the add-on third viral enzyme target on top of a backbone of RdRp inhibition with a nucleoside analog. On the contrary, 2′-O-Mtase activity, while possibly facilitating viral evasion of the host immune response through epigenetic RNA modification, may not be considered crucial for driving the replication cycle relative to other nsps of the RTC. Given no reported inhibitors of 2′-O-Mtase activity in SARS-CoV-2, we posit that this cannot be expected to rise to the significance of either hit 1 or 2 (see graphical abstract). Proteases also are not unique to the Nidovirales order per se; whether they can be of specific 'synthetic lethality' to coronaviridae when inhibited in combination remains to be seen.

Recent clinical trial data somewhat reinforces this doubt whether protease inhibition can be of significant benefit in the context of COVID-19. Lopinavir-ritonavir (protease inhibitor combination approved for HIV) did not produce any clinical benefit over standard of care in hospitalized adult patients with severe COVID-19 [ 51]. Upon reflection, this may not be surprising as lopinavir is designed to be an inhibitor of the retroviral protease which is different from the cysteine protease class of CoVs. While drug repurposing is a time-critical method, this experience should remind us of the fundamental principle of target specificity in preclinical development to enhance clinical success. Encouragingly, recent timely work has determined the crystal structure of SARS-CoV-2 main protease (Mpro) also known as 3CL protease (3CLpro). This in turn allowed the identification of potentially specific protease inhibitors with translational potential through a program of combined structure-based drug design, virtual and high-throughput drug screening [ 52, 53]. It must also be acknowledged that there is no historical precedent for RNA substrate mimetics in translational medicine, which renders them in uncharted waters. Alternatively, small molecule inhibitors of RNaseA have been proposed as a strategy to inhibit coronavirus nsp15 as it bears crystal structural similarity to RnaseA [ 33]. However, as the initial suboptimal efficacy of repurposed HIV protease inhibitors for COVID-19 has demonstrated to some degree, target specificity and substrate affinity must remain paramount even in this brave new world of a pandemic. While RNaseA inhibitors can shut down the endonuclease activity, off target effects in the host would be a concern that needs in vivo evaluation. Therefore, the current in vitro evidence, though limited on RNA substrate mimetics for their potential as specific CoV endonuclease inhibitors, can still serve as the basis for further confirmatory studies to validate the concept.

In conclusion, we hope this insight adds a new layer to a fast expanding armamentarium of therapeutic strategies against COVID-19. Nsp15/NendoU RNA substrate mimetics described in previous studies deserve to be revisited for preclinical optimization and testing, perhaps in combination with nucleoside analogs [ 36].

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

References

1. Senanayake SL. Drug repurposing strategies for COVID-19. Future Drug Discov. 2(2), FDD40 (2020). [ Google Scholar]

43. Kim Y, Jedrzejczak R, Maltseva NI. et al. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

46. Bao L, Deng W, Gao H. et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

48. Farkas C, Fuentes-Villalobos F, Garrido JL, Haigh JJ, Barría MI. Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

49. Thanh Le T, Andreadakis Z, Kumar A. et al. The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

52. Jin Z, Du X, Xu Y. et al. Structure of M[ pro from COVID-19 virus and discovery of its inhibitors. Nature (2020) (Epub ahead of print). Google Scholar]

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COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline.

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Abstract

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a 'double-hit' whereby two CoV-unique protein elements of the replicase-transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the 'double-hit hypothesis'.

Keywords: : combination antiviral therapy, COVID-19, double-hit hypothesis, nsp15/NendoU inhibition, RNA substrate mimetics

Graphical abstract

Replicase-transcriptase complex of SARS-CoV-2 & 'double-hit hypothesis' (Key: CoV-coronavirus, PLpro-Papain-like protease, 3CLpro-3CL protease, RdRp-RNA-dependent RNA polymerase, NendoU-endoribonuclease, ExoN exoribonuclease, 2′-O-Mtase-2′-O-ribose methyltransferase)

As of early May 2020, there is no specific anti-COVID-19 therapy available (clinical management of severe acute respiratory infection when COVID-19 disease is suspected; WHO interim guidance version 1.2). We, among others, have posited that a combination strategy with 'repurposed drugs' could offer the best chance of combating the current COVID-19 pandemic gripping the world [ 1]. In other words, 'multiple drugs versus one virus' where different nodes of the virus-host interactome are simultaneously targeted with antivirals to achieve a synergistic effect [ 2]. Indeed, the case for combination antiviral therapy is becoming increasingly clear, as demonstrated by the modest clinical efficacy hitherto reported of monotherapy, including for the leading RdRp inhibitor remdesivir [ 3].

Such a strategy is not without historical precedence in modern medicine, even before the era of 'personalized medicine', the latter best exemplified by precision oncology. For example, HIV causing AIDS rapidly emerged into the global scene in a very similar pandemic-like fashion during the late 1980s and 1990s. The ensuing decade saw the utilization of this combination strategy with highly active antiretroviral therapy against the new HIV virus. The success of this is evident and AIDS is now considered a manageable, chronic condition if addressed early on in its natural history. As the scientific community scrambles to find a therapeutic solution to face the latest global viral threat in the form of SARS-CoV-2, it is prudent to remember this history's lesson and attempt to replicate it. As such, what could be novel approaches to realize combination therapy in the context of COVID-19?

Rationale for the double-hit hypothesis

To appreciate the multiple drugs versus one virus approach, we must first understand the dynamics of the 'host-virus protein-protein interactome'. Using integrative meta-analysis, Bösl et al. have demonstrated a glimpse into viral evasion mechanisms that target central nodes of the host-virus interactome [ 2]. Here, a seemingly 'hub-and-spoke model' of the host-virus protein-protein interactome emerges in which viruses across families and strains ubiquitously target core host proteins involved in multifunctional cellular pathways for maximal effect. This is of particular importance to smaller RNA viruses (as compared with DNA viruses) with more reliance on host cellular machinery to partly compensate for their higher mutation rate per strand copying [ 4]. RNA viruses such as SARS-CoV-2 rely on RNA-dependent RNA polymerases (RdRps) for faithful transmission of their genomes from host to host [ 5]. However, RdRps are known for their low fidelity of replication, resulting in an elevated rate of spontaneous mutations in RNA viruses [ 6]. Consequently, nucleoside analogs have become a backbone of modern antiviral therapy, exploiting base misincorporation into RNA viral genomes for their therapeutic effect.

Ribavirin is a nucleoside analog broad-spectrum antiviral agent (BSAA) with activity against a number of RNA and DNA viruses [ 7]. It is one of the longest standing antiviral agents included in the WHO's Model List of Essential Medicines (21st list, 2019). These rather promiscuous antiviral properties of ribavirin have been explained by at least five mechanisms; indirect mechanisms, including inosine monophosphate dehydrogenase inhibition affecting the cytoplasmic nucleotide pool and immunomodulatory properties, and direct mechanisms, including RNA capping interference, canonical RdRp inhibition and lethal RNA viral mutagenesis causing 'error catastrophe' [ 8, 9]. However, ribavirin has not demonstrated a significant clinical benefit during previous outbreaks of coronaviridae, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), upon meta-analysis [ 10-12]. This is corroborated by ribavirin's IC ₅₀ in the high micromolar range against human CoVs in vitro, which currently limits its use in this setting due to toxicity at pharmacologically potent doses [ 13, 14]. Notably, this is in contrast to ribavirin's efficacy particularly in combination therapy against a wide variety of viral infections including hepatitis C virus, respiratory syncytial virus and Lassa fever [ 15-17].

Why did a model BSAA with a repertoire of antiviral mechanisms against a number of RNA viruses fail in the face of previous human coronavirus respiratory syndromes? Briefly, work of Ferron et al. using SARS-CoV-1 offers an explanation how a coronavirus can overcome lethal mutagenesis with an endowed exonuclease (ExoN) activity by RNA nucleotide mismatch correction [ 18]. Albeit in vitro evidence, such observations may explain the proven efficacy of a broad-spectrum nucleoside analog against hepatitis C virus without any exonuclease activity and lack of efficacy against SARS or MERS in possession of exonucleases. However, it is perhaps important that we reassess to explore any other overlooked strategies that could potentiate the efficacy of a BSAA in general.

Extrapolating from SARS-CoV-1 & MERS-CoV

Rapid sequencing efforts have established that SARS-CoV-2 is approximately 80% identical to SARS-CoV-1 at the whole genome level [ 19, 20]. This similarity apparently extends to the pathophysiological level of viral replication cycle to some degree, where SARS-CoV-2 shares the same angiotensin-converting enzyme 2 cellular receptor for entry and primes its spike (S) protein with the transmembrane protease serine 2 [ 19, 21-23]. The viral genome can be broadly segmented into regions consisting of nonstructural, structural and accessory genes. Importantly, homology modeling of four key viral proteins (papain-like protease, 3CL protease, helicase and RdRps) suggest that active sites of these viral enzymes to be highly conserved across the three known human coronaviruses (CoVs) with pandemic potential [ 24, 25]. As such, it is a reasonable inference to extrapolate the pharmacology of antiviral agents previously tested against SARS and MERS to rationalize therapeutic options for COVID-19.

Out of the three viral gene regions, those encoding the 16 nonstructural proteins (nsps) perhaps reign supreme in the hierarchy of critical importance to the viral life cycle, as they function in the interphase between cellular entry and exit facilitating ongoing transmission of the virus. Many of these nsps assemble into a supercomplex known as the replicase-transcriptase complex (RTC) with multienzymatic properties that is critical for replication and transcription of subgenomic RNAs [ 26]. Even more unique to CoVs are the nsp15-NendoU and nsp14-ExoN ribonucleases (the former in particular), which can be considered as genetic markers of the Nidovirales order, distinguishing it from all other RNA viruses [ 18, 27]. The work of Ivanov et al. in 2004 following the SARS outbreak perhaps best exemplified the indispensability of nsp15 to a human CoV. A single-nucleotide mutation of nsp15 abolished its endonucleolytic activity and viral RNA synthesis. This observation has been corroborated further in vivo where CoVs expressing mutant nsp15 forms resulted in early robust induction of interferon, apoptosis of macrophages and stimulated a protective immune response leading to significantly attenuated disease in murine models [ 28]. Therefore, this endonuclease can be considered an Achilles' heel-type weaknesses and specific antiviral target [ 29]. However, other studies have indicated that the endoribonuclease activity of CoV nsp15 can be dispensable for the viral replication cycle, albeit required for optimal infection [ 30, 31]. In fact, the key contribution of this evolutionarily conserved endoribonuclease may be the efficient evasion of the innate immune response at the very site of RNA synthesis during the early phase of infection [ 28, 32]. By functioning as an interferon antagonist and evader of double-stranded RNA sensors in macrophages, nsp15 likely represents a primary strategy employed by a CoV to evade the intrinsic antiviral responses and gain a foothold in the host [ 33].

The S protein on the contrary, while crucial for initial viral entry into target cells, has been observed in some CoVs to directly transit to the cell surface without being assembled into virions prior to exocytosis [ 34]. There it mediates fusion between infected and uninfected cells leading to the formation of giant, multinucleated cells facilitating almost a 'paracrine mode of viral spread' in a target tissue. If this is a mechanism faithfully employed by SARS-CoV-2, there could be significant ramification for vaccine development, which represents a major focus of current global efforts. The S protein has intuitively received most attention as the best target for development of monoclonal antibodies. However, the aforementioned mechanism of viral spread could become implicated in circumventing detection and neutralization by antibodies.

Case for inhibiting nsp15/NendoU

The endoribonuclease activity of nsp15/NendoU involves uridylate-specific cleavage of RNAs [ 29, 35-37]. Function of the CoV endonuclease in relation to its viral life cycle has remained somewhat elusive. Evidence exists in animal models for an immunomodulating property during early viral infection in addition to its canonical endoribonuclease activity. Nsp 15 (among other nsps) seems to play a dominant role in suppressing the type I IFN (IFN-α/β)-associated innate immune response by infecting macrophages, thus avoiding detection of viral mRNA by double-stranded RNA sensors [ 28, 32, 38]. Evidence from SARS animal models indicates that it is a delayed yet exuberant IFN-I signaling with an accumulation of neutrophils, monocytes and macrophages in lungs that is the major precipitant of lung immunopathology [ 39]. It is suggested that this temporally dysregulated and late-enhanced innate immune response with an influx of myeloid cells preceding a 'cytokine storm of proinflammatory mediators' drives acute respiratory distress syndrome, which is the dominant cause of COVID-19 mortality [ 40]. Site-directed mutagenesis of the homologous NendoU domain in equine arterivirus (type of nidovirus) has demonstrated up to five log reduction in viral replication and subgenomic RNA accumulation [ 41]. Being an endonuclease and a potential 'suicide enzyme' for an RNA virus, NendoU activity thus is evidently under tight control during replication [ 42]. In toto, nsp15 can be considered a highly specific and unique target for anti-CoV therapeutics.

RNA mimetics as decoy inhibitory ligands against nsp15/NendoU

A high-resolution crystal structure by x-ray diffraction for the nsp15/NendoU of SARS-CoV-2 has now been preliminarily reported leading to some crucial observations [ 43]. In their Protein Data Bank entry of SARS-CoV-2 nsp15/NendoU (PDB ID: 6VWW), Kim et al. report 88% sequence identity and 95% similarity with its closest known homolog in SARS-CoV-1. MERS-CoV nsp15/NendoU, on the contrary, is somewhat more distant with only 50% sequence identity and approximately 65% similarity. More importantly, six key residues forming the catalytic site (His235, His250, Lys290, Thr341, Tyr343 and Ser294) are universally conserved among SARS-CoV-2, SARS-CoV-1 and MERS-CoV, both sequentially and conformationally. This suggests that all three homologs should have extremely similar if not identical substrate affinity. The functionally active form of nsp15 is a double-ring hexamer made of a dimer of trimers, which is consistent with structures of SARS-CoV-1 and MERS-CoV nsp15s [ 43]. Up to six preferentially single-stranded RNAs can engage the hexamer from top and bottom for cleavage (). Therefore, inhibitors of SARS-CoV-1 nsp15 can be expected to inhibit its homolog in SARS-CoV-2. It must be noted that the work of Ivanov et al. [ 29], cited here for primarily supporting nsp15 as a unique nidoviral genetic marker, employed a set of mutant derivatives of HCoV-229E NendoU with Ala substitutions of conserved residues across XendoU/NendoU family. HCoV-229E is a related human CoV which is known to cause common colds and falls in the Alphacoronavirus genus; this contrasts with the Betacoronavirus genus of SARS-CoV-2. As the new nsp15/NendoU crystallographic data from Kim et al. demonstrates, high sequence identity or similarity cannot be expected between HCoV-229E and SARS-CoV-2 given the evolutionary distance [ 43]. Therefore, direct comparison of the active site residues from these studies is difficult and thus necessitates site-directed mutagenesis studies with SARS-CoV-2.

This brings us back to previous work by Bhardwaj et al. in 2006 [ 36]. Using SARS-CoV in vitro models, it was demonstrated that nsp15 specifically cleaves 3′ of uridylate in 16-nt, favorably modified RNA sequences. In a series of elegantly designed experiments, derivatives of the 16-nt prototype RNA substrate (named R16.4) that cannot be efficiently cleaved by nsp15 could still act as competitive inhibitors of this enzyme. These derivatives (e.g., PT13, mR16.4) obtained by minor modifications to the prototype R16.4 sequence (5′GAAGCGAAACCCUAAG3′) retain the ability to interact with nsp15, thus acting as competitive inhibitors of NendoU activity. Single-stranded RNA oligonucleotides with multiple, unpaired uridylate bases had preferentially higher affinity binding to nsp15.

This raises the possibility of a novel approach where RNA substrate mimetics of nsp15 can be deployed as endonuclease inhibitors. An important question is whether combining such endonuclease inhibitors with nucleoside analogs (as backbone antiviral agents) would prove to have an additive or synergistic effect against CoVs. A further possibility is a 'triple-therapy regimen' combining two approved nucleoside analogs, for example ribavirin and sofosbuvir, a combination shown to have tight RdRp inhibition in a recent molecular docking study [ 44].

Future perspective

The leading edge of current global efforts to curb the COVID-19 pandemic is understandably the development of a vaccine. The WHO reported in April 2020, that over 70 candidate vaccines are at different stages of the translational process at an unprecedented speed. Indeed, a proof of principle has been demonstrated in a rhesus macaque model where recovery from infection with SARS-CoV-2 conferred immunity to rechallenge with the same strain [ 45, 46]. However, recent phylogenetic network analyses on reported SARS-CoV-2 genomic samples from around the world have found at least three central variants (A, B, C) indicating in human clonal evolution with possibly different lethality [ 45, 47, 48]. First, most present vaccine candidates have a specific target epitope of SARS-CoV-2, and such rapid antigenic drift of the virus could seriously jeopardize effective vaccination. Second, even an effective preventive vaccine could only perhaps reduce the global infection rate to a basal level, meaning likely cyclical re-emergence of outbreaks similar to seasonal influenza in the near future. This poses a provocative question: is a situation in which vaccine composition has to be constantly changed to suit the dominant virulent strains practically feasible or desirable? All in all, this further highlights the critical role of designing novel therapeutic strategies with antivirals.

Here, we have attempted to elaborate on the previous proposal for using a BSAA combination therapy for COVID-19. Our focus was a concept for further investigation, in other words utilizing already licensed BSAAs with some activity against SARS-CoV-2 concurrently with experimental RNA substrate mimetics as nsp15/NendoU inhibitors. To our knowledge, only in vitro evidence exists for such nuclease inhibitors to-date [ 36]. More lead optimization work on such RNA substrate mimetics to further establish proof of principle is now warranted at a minimum. These can include parallel cellular assays to characterize their stability within microsomes or nanoparticles and off-target properties. As such, the 'double-hit effect' is hypothesis-generating at present and additional supporting data would be necessary prior to advancement of this strategy into in vivo animal model studies and subsequent first-in-human clinical trials. Conversely, the CoV nsp14 would be an attractive target to combine with a BSAA; inhibition of the ExoN catalytic site can be expected to knock out this viral RNA proofreader, thus restoring the potency particularly of nucleoside analogs that are readily available antiviral agents [ 1, 18, 42, 47]. Unlike for NendoU, we are not yet aware of any reported inhibitor class specifically of the CoV exonuclease activity, which limits advancing this combination strategy for now.

We can only speculate on the challenges of RNA substrate mimetics vis-à-vis their translational potential. While the PK/PD profiles of long-standing nucleoside analogs are well established, the in vivo dynamics of potential RNA substrate mimetics are essentially unknown. Additionally, the optimal method or vehicle for their delivery remains to be determined. One possibility is the emerging RNA-based delivery system which can offer significant advantages in terms of simpler design and flexibility of manipulation in comparison to the more traditional viral subunit/vector-driven methods [ 49]. It is also noteworthy that mRNA is under investigation as a robust platform for at least one of the current leading vaccine candidates (mRNA-1273 by Moderna, Inc., MA, USA) which has rapidly been translated into Phase I clinical trial (ClinicalTrials.gov Identifier: NCT04283461). This involves a novel lipid nanoparticle-encapsulated mRNA formulation that encodes for the full-length S protein of SARS-CoV-2 which can be readily administered intramuscularly. In principle, given the relative ease of manufacturing RNA oligonucleotides compared with large recombinant viral proteins, we can anticipate quick adaptability of a similar delivery system to RNA substrate mimetics as cargo. Another important consideration is the temporality of administration of such antiviral therapy. As has been empirically noted, an initially dampened host innate immune response preceding a late exuberant cytokine storm are the hallmarks of severe COVID-19 immunopathology and associated mortality [ 50]. Given the current understanding of CoV-nsp15/NendoU as a major immunomodulator, counteracting viral evasion of host innate immunity in the preliminary stages of disease seems logical. Additionally, formation of double membrane vesicles and RTC activity dominate early during the coronavirus replication cycle before viral protein synthesis escalates to sufficient levels causing viremia and systemic infection. Therefore, the ideal time for inhibition of SARS-CoV-2 endonuclease activity within a combination strategy is perhaps quite early on in COVID-19.

Further, even if favorable PK/PD data are obtained through preclinical testing, the toxicity profiles of such therapeutics solo and in combination for drug-drug interactions would need to be rigorously evaluated in model in vivo systems. The 'double-hit hypothesis' may also dictate that SARS-CoV-2 proteases (i.e., PLpro and 3CLpro) could serve as the add-on third viral enzyme target on top of a backbone of RdRp inhibition with a nucleoside analog. On the contrary, 2′-O-Mtase activity, while possibly facilitating viral evasion of the host immune response through epigenetic RNA modification, may not be considered crucial for driving the replication cycle relative to other nsps of the RTC. Given no reported inhibitors of 2′-O-Mtase activity in SARS-CoV-2, we posit that this cannot be expected to rise to the significance of either hit 1 or 2 (see graphical abstract). Proteases also are not unique to the Nidovirales order per se; whether they can be of specific 'synthetic lethality' to coronaviridae when inhibited in combination remains to be seen.

Recent clinical trial data somewhat reinforces this doubt whether protease inhibition can be of significant benefit in the context of COVID-19. Lopinavir-ritonavir (protease inhibitor combination approved for HIV) did not produce any clinical benefit over standard of care in hospitalized adult patients with severe COVID-19 [ 51]. Upon reflection, this may not be surprising as lopinavir is designed to be an inhibitor of the retroviral protease which is different from the cysteine protease class of CoVs. While drug repurposing is a time-critical method, this experience should remind us of the fundamental principle of target specificity in preclinical development to enhance clinical success. Encouragingly, recent timely work has determined the crystal structure of SARS-CoV-2 main protease (Mpro) also known as 3CL protease (3CLpro). This in turn allowed the identification of potentially specific protease inhibitors with translational potential through a program of combined structure-based drug design, virtual and high-throughput drug screening [ 52, 53]. It must also be acknowledged that there is no historical precedent for RNA substrate mimetics in translational medicine, which renders them in uncharted waters. Alternatively, small molecule inhibitors of RNaseA have been proposed as a strategy to inhibit coronavirus nsp15 as it bears crystal structural similarity to RnaseA [ 33]. However, as the initial suboptimal efficacy of repurposed HIV protease inhibitors for COVID-19 has demonstrated to some degree, target specificity and substrate affinity must remain paramount even in this brave new world of a pandemic. While RNaseA inhibitors can shut down the endonuclease activity, off target effects in the host would be a concern that needs in vivo evaluation. Therefore, the current in vitro evidence, though limited on RNA substrate mimetics for their potential as specific CoV endonuclease inhibitors, can still serve as the basis for further confirmatory studies to validate the concept.

In conclusion, we hope this insight adds a new layer to a fast expanding armamentarium of therapeutic strategies against COVID-19. Nsp15/NendoU RNA substrate mimetics described in previous studies deserve to be revisited for preclinical optimization and testing, perhaps in combination with nucleoside analogs [ 36].

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

References

1. Senanayake SL. Drug repurposing strategies for COVID-19. Future Drug Discov. 2(2), FDD40 (2020). [ Google Scholar]

43. Kim Y, Jedrzejczak R, Maltseva NI. et al. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

46. Bao L, Deng W, Gao H. et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

48. Farkas C, Fuentes-Villalobos F, Garrido JL, Haigh JJ, Barría MI. Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

49. Thanh Le T, Andreadakis Z, Kumar A. et al. The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

52. Jin Z, Du X, Xu Y. et al. Structure of M[ pro from COVID-19 virus and discovery of its inhibitors. Nature (2020) (Epub ahead of print). Google Scholar]

# data[].attributes.attachment_link.description
COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline.

# data[].attributes.attachment_link.readable_text
Abstract

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a 'double-hit' whereby two CoV-unique protein elements of the replicase-transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the 'double-hit hypothesis'.

Keywords: : combination antiviral therapy, COVID-19, double-hit hypothesis, nsp15/NendoU inhibition, RNA substrate mimetics

Graphical abstract

Replicase-transcriptase complex of SARS-CoV-2 & 'double-hit hypothesis' (Key: CoV-coronavirus, PLpro-Papain-like protease, 3CLpro-3CL protease, RdRp-RNA-dependent RNA polymerase, NendoU-endoribonuclease, ExoN exoribonuclease, 2′-O-Mtase-2′-O-ribose methyltransferase)

As of early May 2020, there is no specific anti-COVID-19 therapy available (clinical management of severe acute respiratory infection when COVID-19 disease is suspected; WHO interim guidance version 1.2). We, among others, have posited that a combination strategy with 'repurposed drugs' could offer the best chance of combating the current COVID-19 pandemic gripping the world [ 1]. In other words, 'multiple drugs versus one virus' where different nodes of the virus-host interactome are simultaneously targeted with antivirals to achieve a synergistic effect [ 2]. Indeed, the case for combination antiviral therapy is becoming increasingly clear, as demonstrated by the modest clinical efficacy hitherto reported of monotherapy, including for the leading RdRp inhibitor remdesivir [ 3].

Such a strategy is not without historical precedence in modern medicine, even before the era of 'personalized medicine', the latter best exemplified by precision oncology. For example, HIV causing AIDS rapidly emerged into the global scene in a very similar pandemic-like fashion during the late 1980s and 1990s. The ensuing decade saw the utilization of this combination strategy with highly active antiretroviral therapy against the new HIV virus. The success of this is evident and AIDS is now considered a manageable, chronic condition if addressed early on in its natural history. As the scientific community scrambles to find a therapeutic solution to face the latest global viral threat in the form of SARS-CoV-2, it is prudent to remember this history's lesson and attempt to replicate it. As such, what could be novel approaches to realize combination therapy in the context of COVID-19?

Rationale for the double-hit hypothesis

To appreciate the multiple drugs versus one virus approach, we must first understand the dynamics of the 'host-virus protein-protein interactome'. Using integrative meta-analysis, Bösl et al. have demonstrated a glimpse into viral evasion mechanisms that target central nodes of the host-virus interactome [ 2]. Here, a seemingly 'hub-and-spoke model' of the host-virus protein-protein interactome emerges in which viruses across families and strains ubiquitously target core host proteins involved in multifunctional cellular pathways for maximal effect. This is of particular importance to smaller RNA viruses (as compared with DNA viruses) with more reliance on host cellular machinery to partly compensate for their higher mutation rate per strand copying [ 4]. RNA viruses such as SARS-CoV-2 rely on RNA-dependent RNA polymerases (RdRps) for faithful transmission of their genomes from host to host [ 5]. However, RdRps are known for their low fidelity of replication, resulting in an elevated rate of spontaneous mutations in RNA viruses [ 6]. Consequently, nucleoside analogs have become a backbone of modern antiviral therapy, exploiting base misincorporation into RNA viral genomes for their therapeutic effect.

Ribavirin is a nucleoside analog broad-spectrum antiviral agent (BSAA) with activity against a number of RNA and DNA viruses [ 7]. It is one of the longest standing antiviral agents included in the WHO's Model List of Essential Medicines (21st list, 2019). These rather promiscuous antiviral properties of ribavirin have been explained by at least five mechanisms; indirect mechanisms, including inosine monophosphate dehydrogenase inhibition affecting the cytoplasmic nucleotide pool and immunomodulatory properties, and direct mechanisms, including RNA capping interference, canonical RdRp inhibition and lethal RNA viral mutagenesis causing 'error catastrophe' [ 8, 9]. However, ribavirin has not demonstrated a significant clinical benefit during previous outbreaks of coronaviridae, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), upon meta-analysis [ 10-12]. This is corroborated by ribavirin's IC ₅₀ in the high micromolar range against human CoVs in vitro, which currently limits its use in this setting due to toxicity at pharmacologically potent doses [ 13, 14]. Notably, this is in contrast to ribavirin's efficacy particularly in combination therapy against a wide variety of viral infections including hepatitis C virus, respiratory syncytial virus and Lassa fever [ 15-17].

Why did a model BSAA with a repertoire of antiviral mechanisms against a number of RNA viruses fail in the face of previous human coronavirus respiratory syndromes? Briefly, work of Ferron et al. using SARS-CoV-1 offers an explanation how a coronavirus can overcome lethal mutagenesis with an endowed exonuclease (ExoN) activity by RNA nucleotide mismatch correction [ 18]. Albeit in vitro evidence, such observations may explain the proven efficacy of a broad-spectrum nucleoside analog against hepatitis C virus without any exonuclease activity and lack of efficacy against SARS or MERS in possession of exonucleases. However, it is perhaps important that we reassess to explore any other overlooked strategies that could potentiate the efficacy of a BSAA in general.

Extrapolating from SARS-CoV-1 & MERS-CoV

Rapid sequencing efforts have established that SARS-CoV-2 is approximately 80% identical to SARS-CoV-1 at the whole genome level [ 19, 20]. This similarity apparently extends to the pathophysiological level of viral replication cycle to some degree, where SARS-CoV-2 shares the same angiotensin-converting enzyme 2 cellular receptor for entry and primes its spike (S) protein with the transmembrane protease serine 2 [ 19, 21-23]. The viral genome can be broadly segmented into regions consisting of nonstructural, structural and accessory genes. Importantly, homology modeling of four key viral proteins (papain-like protease, 3CL protease, helicase and RdRps) suggest that active sites of these viral enzymes to be highly conserved across the three known human coronaviruses (CoVs) with pandemic potential [ 24, 25]. As such, it is a reasonable inference to extrapolate the pharmacology of antiviral agents previously tested against SARS and MERS to rationalize therapeutic options for COVID-19.

Out of the three viral gene regions, those encoding the 16 nonstructural proteins (nsps) perhaps reign supreme in the hierarchy of critical importance to the viral life cycle, as they function in the interphase between cellular entry and exit facilitating ongoing transmission of the virus. Many of these nsps assemble into a supercomplex known as the replicase-transcriptase complex (RTC) with multienzymatic properties that is critical for replication and transcription of subgenomic RNAs [ 26]. Even more unique to CoVs are the nsp15-NendoU and nsp14-ExoN ribonucleases (the former in particular), which can be considered as genetic markers of the Nidovirales order, distinguishing it from all other RNA viruses [ 18, 27]. The work of Ivanov et al. in 2004 following the SARS outbreak perhaps best exemplified the indispensability of nsp15 to a human CoV. A single-nucleotide mutation of nsp15 abolished its endonucleolytic activity and viral RNA synthesis. This observation has been corroborated further in vivo where CoVs expressing mutant nsp15 forms resulted in early robust induction of interferon, apoptosis of macrophages and stimulated a protective immune response leading to significantly attenuated disease in murine models [ 28]. Therefore, this endonuclease can be considered an Achilles' heel-type weaknesses and specific antiviral target [ 29]. However, other studies have indicated that the endoribonuclease activity of CoV nsp15 can be dispensable for the viral replication cycle, albeit required for optimal infection [ 30, 31]. In fact, the key contribution of this evolutionarily conserved endoribonuclease may be the efficient evasion of the innate immune response at the very site of RNA synthesis during the early phase of infection [ 28, 32]. By functioning as an interferon antagonist and evader of double-stranded RNA sensors in macrophages, nsp15 likely represents a primary strategy employed by a CoV to evade the intrinsic antiviral responses and gain a foothold in the host [ 33].

The S protein on the contrary, while crucial for initial viral entry into target cells, has been observed in some CoVs to directly transit to the cell surface without being assembled into virions prior to exocytosis [ 34]. There it mediates fusion between infected and uninfected cells leading to the formation of giant, multinucleated cells facilitating almost a 'paracrine mode of viral spread' in a target tissue. If this is a mechanism faithfully employed by SARS-CoV-2, there could be significant ramification for vaccine development, which represents a major focus of current global efforts. The S protein has intuitively received most attention as the best target for development of monoclonal antibodies. However, the aforementioned mechanism of viral spread could become implicated in circumventing detection and neutralization by antibodies.

Case for inhibiting nsp15/NendoU

The endoribonuclease activity of nsp15/NendoU involves uridylate-specific cleavage of RNAs [ 29, 35-37]. Function of the CoV endonuclease in relation to its viral life cycle has remained somewhat elusive. Evidence exists in animal models for an immunomodulating property during early viral infection in addition to its canonical endoribonuclease activity. Nsp 15 (among other nsps) seems to play a dominant role in suppressing the type I IFN (IFN-α/β)-associated innate immune response by infecting macrophages, thus avoiding detection of viral mRNA by double-stranded RNA sensors [ 28, 32, 38]. Evidence from SARS animal models indicates that it is a delayed yet exuberant IFN-I signaling with an accumulation of neutrophils, monocytes and macrophages in lungs that is the major precipitant of lung immunopathology [ 39]. It is suggested that this temporally dysregulated and late-enhanced innate immune response with an influx of myeloid cells preceding a 'cytokine storm of proinflammatory mediators' drives acute respiratory distress syndrome, which is the dominant cause of COVID-19 mortality [ 40]. Site-directed mutagenesis of the homologous NendoU domain in equine arterivirus (type of nidovirus) has demonstrated up to five log reduction in viral replication and subgenomic RNA accumulation [ 41]. Being an endonuclease and a potential 'suicide enzyme' for an RNA virus, NendoU activity thus is evidently under tight control during replication [ 42]. In toto, nsp15 can be considered a highly specific and unique target for anti-CoV therapeutics.

RNA mimetics as decoy inhibitory ligands against nsp15/NendoU

A high-resolution crystal structure by x-ray diffraction for the nsp15/NendoU of SARS-CoV-2 has now been preliminarily reported leading to some crucial observations [ 43]. In their Protein Data Bank entry of SARS-CoV-2 nsp15/NendoU (PDB ID: 6VWW), Kim et al. report 88% sequence identity and 95% similarity with its closest known homolog in SARS-CoV-1. MERS-CoV nsp15/NendoU, on the contrary, is somewhat more distant with only 50% sequence identity and approximately 65% similarity. More importantly, six key residues forming the catalytic site (His235, His250, Lys290, Thr341, Tyr343 and Ser294) are universally conserved among SARS-CoV-2, SARS-CoV-1 and MERS-CoV, both sequentially and conformationally. This suggests that all three homologs should have extremely similar if not identical substrate affinity. The functionally active form of nsp15 is a double-ring hexamer made of a dimer of trimers, which is consistent with structures of SARS-CoV-1 and MERS-CoV nsp15s [ 43]. Up to six preferentially single-stranded RNAs can engage the hexamer from top and bottom for cleavage (). Therefore, inhibitors of SARS-CoV-1 nsp15 can be expected to inhibit its homolog in SARS-CoV-2. It must be noted that the work of Ivanov et al. [ 29], cited here for primarily supporting nsp15 as a unique nidoviral genetic marker, employed a set of mutant derivatives of HCoV-229E NendoU with Ala substitutions of conserved residues across XendoU/NendoU family. HCoV-229E is a related human CoV which is known to cause common colds and falls in the Alphacoronavirus genus; this contrasts with the Betacoronavirus genus of SARS-CoV-2. As the new nsp15/NendoU crystallographic data from Kim et al. demonstrates, high sequence identity or similarity cannot be expected between HCoV-229E and SARS-CoV-2 given the evolutionary distance [ 43]. Therefore, direct comparison of the active site residues from these studies is difficult and thus necessitates site-directed mutagenesis studies with SARS-CoV-2.

This brings us back to previous work by Bhardwaj et al. in 2006 [ 36]. Using SARS-CoV in vitro models, it was demonstrated that nsp15 specifically cleaves 3′ of uridylate in 16-nt, favorably modified RNA sequences. In a series of elegantly designed experiments, derivatives of the 16-nt prototype RNA substrate (named R16.4) that cannot be efficiently cleaved by nsp15 could still act as competitive inhibitors of this enzyme. These derivatives (e.g., PT13, mR16.4) obtained by minor modifications to the prototype R16.4 sequence (5′GAAGCGAAACCCUAAG3′) retain the ability to interact with nsp15, thus acting as competitive inhibitors of NendoU activity. Single-stranded RNA oligonucleotides with multiple, unpaired uridylate bases had preferentially higher affinity binding to nsp15.

This raises the possibility of a novel approach where RNA substrate mimetics of nsp15 can be deployed as endonuclease inhibitors. An important question is whether combining such endonuclease inhibitors with nucleoside analogs (as backbone antiviral agents) would prove to have an additive or synergistic effect against CoVs. A further possibility is a 'triple-therapy regimen' combining two approved nucleoside analogs, for example ribavirin and sofosbuvir, a combination shown to have tight RdRp inhibition in a recent molecular docking study [ 44].

Future perspective

The leading edge of current global efforts to curb the COVID-19 pandemic is understandably the development of a vaccine. The WHO reported in April 2020, that over 70 candidate vaccines are at different stages of the translational process at an unprecedented speed. Indeed, a proof of principle has been demonstrated in a rhesus macaque model where recovery from infection with SARS-CoV-2 conferred immunity to rechallenge with the same strain [ 45, 46]. However, recent phylogenetic network analyses on reported SARS-CoV-2 genomic samples from around the world have found at least three central variants (A, B, C) indicating in human clonal evolution with possibly different lethality [ 45, 47, 48]. First, most present vaccine candidates have a specific target epitope of SARS-CoV-2, and such rapid antigenic drift of the virus could seriously jeopardize effective vaccination. Second, even an effective preventive vaccine could only perhaps reduce the global infection rate to a basal level, meaning likely cyclical re-emergence of outbreaks similar to seasonal influenza in the near future. This poses a provocative question: is a situation in which vaccine composition has to be constantly changed to suit the dominant virulent strains practically feasible or desirable? All in all, this further highlights the critical role of designing novel therapeutic strategies with antivirals.

Here, we have attempted to elaborate on the previous proposal for using a BSAA combination therapy for COVID-19. Our focus was a concept for further investigation, in other words utilizing already licensed BSAAs with some activity against SARS-CoV-2 concurrently with experimental RNA substrate mimetics as nsp15/NendoU inhibitors. To our knowledge, only in vitro evidence exists for such nuclease inhibitors to-date [ 36]. More lead optimization work on such RNA substrate mimetics to further establish proof of principle is now warranted at a minimum. These can include parallel cellular assays to characterize their stability within microsomes or nanoparticles and off-target properties. As such, the 'double-hit effect' is hypothesis-generating at present and additional supporting data would be necessary prior to advancement of this strategy into in vivo animal model studies and subsequent first-in-human clinical trials. Conversely, the CoV nsp14 would be an attractive target to combine with a BSAA; inhibition of the ExoN catalytic site can be expected to knock out this viral RNA proofreader, thus restoring the potency particularly of nucleoside analogs that are readily available antiviral agents [ 1, 18, 42, 47]. Unlike for NendoU, we are not yet aware of any reported inhibitor class specifically of the CoV exonuclease activity, which limits advancing this combination strategy for now.

We can only speculate on the challenges of RNA substrate mimetics vis-à-vis their translational potential. While the PK/PD profiles of long-standing nucleoside analogs are well established, the in vivo dynamics of potential RNA substrate mimetics are essentially unknown. Additionally, the optimal method or vehicle for their delivery remains to be determined. One possibility is the emerging RNA-based delivery system which can offer significant advantages in terms of simpler design and flexibility of manipulation in comparison to the more traditional viral subunit/vector-driven methods [ 49]. It is also noteworthy that mRNA is under investigation as a robust platform for at least one of the current leading vaccine candidates (mRNA-1273 by Moderna, Inc., MA, USA) which has rapidly been translated into Phase I clinical trial (ClinicalTrials.gov Identifier: NCT04283461). This involves a novel lipid nanoparticle-encapsulated mRNA formulation that encodes for the full-length S protein of SARS-CoV-2 which can be readily administered intramuscularly. In principle, given the relative ease of manufacturing RNA oligonucleotides compared with large recombinant viral proteins, we can anticipate quick adaptability of a similar delivery system to RNA substrate mimetics as cargo. Another important consideration is the temporality of administration of such antiviral therapy. As has been empirically noted, an initially dampened host innate immune response preceding a late exuberant cytokine storm are the hallmarks of severe COVID-19 immunopathology and associated mortality [ 50]. Given the current understanding of CoV-nsp15/NendoU as a major immunomodulator, counteracting viral evasion of host innate immunity in the preliminary stages of disease seems logical. Additionally, formation of double membrane vesicles and RTC activity dominate early during the coronavirus replication cycle before viral protein synthesis escalates to sufficient levels causing viremia and systemic infection. Therefore, the ideal time for inhibition of SARS-CoV-2 endonuclease activity within a combination strategy is perhaps quite early on in COVID-19.

Further, even if favorable PK/PD data are obtained through preclinical testing, the toxicity profiles of such therapeutics solo and in combination for drug-drug interactions would need to be rigorously evaluated in model in vivo systems. The 'double-hit hypothesis' may also dictate that SARS-CoV-2 proteases (i.e., PLpro and 3CLpro) could serve as the add-on third viral enzyme target on top of a backbone of RdRp inhibition with a nucleoside analog. On the contrary, 2′-O-Mtase activity, while possibly facilitating viral evasion of the host immune response through epigenetic RNA modification, may not be considered crucial for driving the replication cycle relative to other nsps of the RTC. Given no reported inhibitors of 2′-O-Mtase activity in SARS-CoV-2, we posit that this cannot be expected to rise to the significance of either hit 1 or 2 (see graphical abstract). Proteases also are not unique to the Nidovirales order per se; whether they can be of specific 'synthetic lethality' to coronaviridae when inhibited in combination remains to be seen.

Recent clinical trial data somewhat reinforces this doubt whether protease inhibition can be of significant benefit in the context of COVID-19. Lopinavir-ritonavir (protease inhibitor combination approved for HIV) did not produce any clinical benefit over standard of care in hospitalized adult patients with severe COVID-19 [ 51]. Upon reflection, this may not be surprising as lopinavir is designed to be an inhibitor of the retroviral protease which is different from the cysteine protease class of CoVs. While drug repurposing is a time-critical method, this experience should remind us of the fundamental principle of target specificity in preclinical development to enhance clinical success. Encouragingly, recent timely work has determined the crystal structure of SARS-CoV-2 main protease (Mpro) also known as 3CL protease (3CLpro). This in turn allowed the identification of potentially specific protease inhibitors with translational potential through a program of combined structure-based drug design, virtual and high-throughput drug screening [ 52, 53]. It must also be acknowledged that there is no historical precedent for RNA substrate mimetics in translational medicine, which renders them in uncharted waters. Alternatively, small molecule inhibitors of RNaseA have been proposed as a strategy to inhibit coronavirus nsp15 as it bears crystal structural similarity to RnaseA [ 33]. However, as the initial suboptimal efficacy of repurposed HIV protease inhibitors for COVID-19 has demonstrated to some degree, target specificity and substrate affinity must remain paramount even in this brave new world of a pandemic. While RNaseA inhibitors can shut down the endonuclease activity, off target effects in the host would be a concern that needs in vivo evaluation. Therefore, the current in vitro evidence, though limited on RNA substrate mimetics for their potential as specific CoV endonuclease inhibitors, can still serve as the basis for further confirmatory studies to validate the concept.

In conclusion, we hope this insight adds a new layer to a fast expanding armamentarium of therapeutic strategies against COVID-19. Nsp15/NendoU RNA substrate mimetics described in previous studies deserve to be revisited for preclinical optimization and testing, perhaps in combination with nucleoside analogs [ 36].

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

References

1. Senanayake SL. Drug repurposing strategies for COVID-19. Future Drug Discov. 2(2), FDD40 (2020). [ Google Scholar]

43. Kim Y, Jedrzejczak R, Maltseva NI. et al. Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein Sci. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

46. Bao L, Deng W, Gao H. et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

48. Farkas C, Fuentes-Villalobos F, Garrido JL, Haigh JJ, Barría MI. Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

49. Thanh Le T, Andreadakis Z, Kumar A. et al. The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov. (2020) (Epub ahead of print). [[ PubMed] Google Scholar]

52. Jin Z, Du X, Xu Y. et al. Structure of M[ pro from COVID-19 virus and discovery of its inhibitors. Nature (2020) (Epub ahead of print). Google Scholar]

# data[].attributes.attachment_link.description
COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline.

# data[].attributes.attachment_link.readable_text
Abstract

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a 'double-hit' whereby two CoV-unique protein elements of the replicase-transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the 'double-hit hypothesis'.

Keywords: : combination antiviral therapy, COVID-19, double-hit hypothesis, nsp15/NendoU inhibition, RNA substrate mimetics

Graphical abstract

Replicase-transcriptase complex of SARS-CoV-2 & 'double-hit hypothesis' (Key: CoV-coronavirus, PLpro-Papain-like protease, 3CLpro-3CL protease, RdRp-RNA-dependent RNA polymerase, NendoU-endoribonuclease, ExoN exoribonuclease, 2′-O-Mtase-2′-O-ribose methyltransferase)

As of early May 2020, there is no specific anti-COVID-19 therapy available (clinical management of severe acute respiratory infection when COVID-19 disease is suspected; WHO interim guidance version 1.2). We, among others, have posited that a combination strategy with 'repurposed drugs' could offer the best chance of combating the current COVID-19 pandemic gripping the world [ 1]. In other words, 'multiple drugs versus one virus' where different nodes of the virus-host interactome are simultaneously targeted with antivirals to achieve a synergistic effect [ 2]. Indeed, the case for combination antiviral therapy is becoming increasingly clear, as demonstrated by the modest clinical efficacy hitherto reported of monotherapy, including for the leading RdRp inhibitor remdesivir [ 3].

Such a strategy is not without historical precedence in modern medicine, even before the era of 'personalized medicine', the latter best exemplified by precision oncology. For example, HIV causing AIDS rapidly emerged into the global scene in a very similar pandemic-like fashion during the late 1980s and 1990s. The ensuing decade saw the utilization of this combination strategy with highly active antiretroviral therapy against the new HIV virus. The success of this is evident and AIDS is now considered a manageable, chronic condition if addressed early on in its natural history. As the scientific community scrambles to find a therapeutic solution to face the latest global viral threat in the form of SARS-CoV-2, it is prudent to remember this history's lesson and attempt to replicate it. As such, what could be novel approaches to realize combination therapy in the context of COVID-19?

Rationale for the double-hit hypothesis

To appreciate the multiple drugs versus one virus approach, we must first understand the dynamics of the 'host-virus protein-protein interactome'. Using integrative meta-analysis, Bösl et al. have demonstrated a glimpse into viral evasion mechanisms that target central nodes of the host-virus interactome [ 2]. Here, a seemingly 'hub-and-spoke model' of the host-virus protein-protein interactome emerges in which viruses across families and strains ubiquitously target core host proteins involved in multifunctional cellular pathways for maximal effect. This is of particular importance to smaller RNA viruses (as compared with DNA viruses) with more reliance on host cellular machinery to partly compensate for their higher mutation rate per strand copying [ 4]. RNA viruses such as SARS-CoV-2 rely on RNA-dependent RNA polymerases (RdRps) for faithful transmission of their genomes from host to host [ 5]. However, RdRps are known for their low fidelity of replication, resulting in an elevated rate of spontaneous mutations in RNA viruses [ 6]. Consequently, nucleoside analogs have become a backbone of modern antiviral therapy, exploiting base misincorporation into RNA viral genomes for their therapeutic effect.

Ribavirin is a nucleoside analog broad-spectrum antiviral agent (BSAA) with activity against a number of RNA and DNA viruses [ 7]. It is one of the longest standing antiviral agents included in the WHO's Model List of Essential Medicines (21st list, 2019). These rather promiscuous antiviral properties of ribavirin have been explained by at least five mechanisms; indirect mechanisms, including inosine monophosphate dehydrogenase inhibition affecting the cytoplasmic nucleotide pool and immunomodulatory properties, and direct mechanisms, including RNA capping interference, canonical RdRp inhibition and lethal RNA viral mutagenesis causing 'error catastrophe' [ 8, 9]. However, ribavirin has not demonstrated a significant clinical benefit during previous outbreaks of coronaviridae, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), upon meta-analysis [ 10-12]. This is corroborated by ribavirin's IC ₅₀ in the high micromolar range against human CoVs in vitro, which currently limits its use in this setting due to toxicity at pharmacologically potent doses [ 13, 14]. Notably, this is in contrast to ribavirin's efficacy particularly in combination therapy against a wide variety of viral infections including hepatitis C virus, respiratory syncytial virus and Lassa fever [ 15-17].

Why did a model BSAA with a repertoire of antiviral mechanisms against a number of RNA viruses fail in the face of previous human coronavirus respiratory syndromes? Briefly, work of Ferron et al. using SARS-CoV-1 offers an explanation how a coronavirus can overcome lethal mutagenesis with an endowed exonuclease (ExoN) activity by RNA nucleotide mismatch correction [ 18]. Albeit in vitro evidence, such observations may explain the proven efficacy of a broad-spectrum nucleoside analog against hepatitis C virus without any exonuclease activity and lack of efficacy against SARS or MERS in possession of exonucleases. However, it is perhaps important that we reassess to explore any other overlooked strategies that could potentiate the efficacy of a BSAA in general.

Extrapolating from SARS-CoV-1 & MERS-CoV

Rapid sequencing efforts have established that SARS-CoV-2 is approximately 80% identical to SARS-CoV-1 at the whole genome level [ 19, 20]. This similarity apparently extends to the pathophysiological level of viral replication cycle to some degree, where SARS-CoV-2 shares the same angiotensin-converting enzyme 2 cellular receptor for entry and primes its spike (S) protein with the transmembrane protease serine 2 [ 19, 21-23]. The viral genome can be broadly segmented into regions consisting of nonstructural, structural and accessory genes. Importantly, homology modeling of four key viral proteins (papain-like protease, 3CL protease, helicase and RdRps) suggest that active sites of these viral enzymes to be highly conserved across the three known human coronaviruses (CoVs) with pandemic potential [ 24, 25]. As such, it is a reasonable inference to extrapolate the pharmacology of antiviral agents previously tested against SARS and MERS to rationalize therapeutic options for COVID-19.

Out of the three viral gene regions, those encoding the 16 nonstructural proteins (nsps) perhaps reign supreme in the hierarchy of critical importance to the viral life cycle, as they function in the interphase between cellular entry and exit facilitating ongoing transmission of the virus. Many of these nsps assemble into a supercomplex known as the replicase-transcriptase complex (RTC) with multienzymatic properties that is critical for replication and transcription of subgenomic RNAs [ 26]. Even more unique to CoVs are the nsp15-NendoU and nsp14-ExoN ribonucleases (the former in particular), which can be considered as genetic markers of the Nidovirales order, distinguishing it from all other RNA viruses [ 18, 27]. The work of Ivanov et al. in 2004 following the SARS outbreak perhaps best exemplified the indispensability of nsp15 to a human CoV. A single-nucleotide mutation of nsp15 abolished its endonucleolytic activity and viral RNA synthesis. This observation has been corroborated further in vivo where CoVs expressing mutant nsp15 forms resulted in early robust induction of interferon, apoptosis of macrophages and stimulated a protective immune response leading to significantly attenuated disease in murine models [ 28]. Therefore, this endonuclease can be considered an Achilles' heel-type weaknesses and specific antiviral target [ 29]. However, other studies have indicated that the endoribonuclease activity of CoV nsp15 can be dispensable for the viral replication cycle, albeit required for optimal infection [ 30, 31]. In fact, the key contribution of this evolutionarily conserved endoribonuclease may be the efficient evasion of the innate immune response at the very site of RNA synthesis during the early phase of infection [ 28, 32]. By functioning as an interferon antagonist and evader of double-stranded RNA sensors in macrophages, nsp15 likely represents a primary strategy employed by a CoV to evade the intrinsic antiviral responses and gain a foothold in the host [ 33].

The S protein on the contrary, while crucial for initial viral entry into target cells, has been observed in some CoVs to directly transit to the cell surface without being assembled into virions prior to exocytosis [ 34]. There it mediates fusion between infected and uninfected cells leading to the formation of giant, multinucleated cells facilitating almost a 'paracrine mode of viral spread' in a target tissue. If this is a mechanism faithfully employed by SARS-CoV-2, there could be significant ramification for vaccine development, which represents a major focus of current global efforts. The S protein has intuitively received most attention as the best target for development of monoclonal antibodies. However, the aforementioned mechanism of viral spread could become implicated in circumventing detection and neutralization by antibodies.

Case for inhibiting nsp15/NendoU

The endoribonuclease activity of nsp15/NendoU involves uridylate-specific cleavage of RNAs [ 29, 35-37]. Function of the CoV endonuclease in relation to its viral life cycle has remained somewhat elusive. Evidence exists in animal models for an immunomodulating property during early viral infection in addition to its canonical endoribonuclease activity. Nsp 15 (among other nsps) seems to play a dominant role in suppressing the type I IFN (IFN-α/β)-associated innate immune response by infecting macrophages, thus avoiding detection of viral mRNA by double-stranded RNA sensors [ 28, 32, 38]. Evidence from SARS animal models indicates that it is a delayed yet exuberant IFN-I signaling with an accumulation of neutrophils, monocytes and macrophages in lungs that is the major precipitant of lung immunopathology [ 39]. It is suggested that this temporally dysregulated and late-enhanced innate immune response with an influx of myeloid cells preceding a 'cytokine storm of proinflammatory mediators' drives acute respiratory distress syndrome, which is the dominant cause of COVID-19 mortality [ 40]. Site-directed mutagenesis of the homologous NendoU domain in equine arterivirus (type of nidovirus) has demonstrated up to five log reduction in viral replication and subgenomic RNA accumulation [ 41]. Being an endonuclease and a potential 'suicide enzyme' for an RNA virus, NendoU activity thus is evidently under tight control during replication [ 42]. In toto, nsp15 can be considered a highly specific and unique target for anti-CoV therapeutics.

RNA mimetics as decoy inhibitory ligands against nsp15/NendoU

A high-resolution crystal structure by x-ray diffraction for the nsp15/NendoU of SARS-CoV-2 has now been preliminarily reported leading to some crucial observations [ 43]. In their Protein Data Bank entry of SARS-CoV-2 nsp15/NendoU (PDB ID: 6VWW), Kim et al. report 88% sequence identity and 95% similarity with its closest known homolog in SARS-CoV-1. MERS-CoV nsp15/NendoU, on the contrary, is somewhat more distant with only 50% sequence identity and approximately 65% similarity. More importantly, six key residues forming the catalytic site (His235, His250, Lys290, Thr341, Tyr343 and Ser294) are universally conserved among SARS-CoV-2, SARS-CoV-1 and MERS-CoV, both sequentially and conformationally. This suggests that all three homologs should have extremely similar if not identical substrate affinity. The functionally active form of nsp15 is a double-ring hexamer made of a dimer of trimers, which is consistent with structures of SARS-CoV-1 and MERS-CoV nsp15s [ 43]. Up to six preferentially single-stranded RNAs can engage the hexamer from top and bottom for cleavage (). Therefore, inhibitors of SARS-CoV-1 nsp15 can be expected to inhibit its homolog in SARS-CoV-2. It must be noted that the work of Ivanov et al. [ 29], cited here for primarily supporting nsp15 as a unique nidoviral genetic marker, employed a set of mutant derivatives of HCoV-229E NendoU with Ala substitutions of conserved residues across XendoU/NendoU family. HCoV-229E is a related human CoV which is known to cause common colds and falls in the Alphacoronavirus genus; this contrasts with the Betacoronavirus genus of SARS-CoV-2. As the new nsp15/NendoU crystallographic data from Kim et al. demonstrates, high sequence identity or similarity cannot be expected between HCoV-229E and SARS-CoV-2 given the evolutionary distance [ 43]. Therefore, direct comparison of the active site residues from these studies is difficult and thus necessitates site-directed mutagenesis studies with SARS-CoV-2.

This brings us back to previous work by Bhardwaj et al. in 2006 [ 36]. Using SARS-CoV in vitro models, it was demonstrated that nsp15 specifically cleaves 3′ of uridylate in 16-nt, favorably modified RNA sequences. In a series of elegantly designed experiments, derivatives of the 16-nt prototype RNA substrate (named R16.4) that cannot be efficiently cleaved by nsp15 could still act as competitive inhibitors of this enzyme. These derivatives (e.g., PT13, mR16.4) obtained by minor modifications to the prototype R16.4 sequence (5′GAAGCGAAACCCUAAG3′) retain the ability to interact with nsp15, thus acting as competitive inhibitors of NendoU activity. Single-stranded RNA oligonucleotides with multiple, unpaired uridylate bases had preferentially higher affinity binding to nsp15.

This raises the possibility of a novel approach where RNA substrate mimetics of nsp15 can be deployed as endonuclease inhibitors. An important question is whether combining such endonuclease inhibitors with nucleoside analogs (as backbone antiviral agents) would prove to have an additive or synergistic effect against CoVs. A further possibility is a 'triple-therapy regimen' combining two approved nucleoside analogs, for example ribavirin and sofosbuvir, a combination shown to have tight RdRp inhibition in a recent molecular docking study [ 44].

Future perspective

The leading edge of current global efforts to curb the COVID-19 pandemic is understandably the development of a vaccine. The WHO reported in April 2020, that over 70 candidate vaccines are at different stages of the translational process at an unprecedented speed. Indeed, a proof of principle has been demonstrated in a rhesus macaque model where recovery from infection with SARS-CoV-2 conferred immunity to rechallenge with the same strain [ 45, 46]. However, recent phylogenetic network analyses on reported SARS-CoV-2 genomic samples from around the world have found at least three central variants (A, B, C) indicating in human clonal evolution with possibly different lethality [ 45, 47, 48]. First, most present vaccine candidates have a specific target epitope of SARS-CoV-2, and such rapid antigenic drift of the virus could seriously jeopardize effective vaccination. Second, even an effective preventive vaccine could only perhaps reduce the global infection rate to a basal level, meaning likely cyclical re-emergence of outbreaks similar to seasonal influenza in the near future. This poses a provocative question: is a situation in which vaccine composition has to be constantly changed to suit the dominant virulent strains practically feasible or desirable? All in all, this further highlights the critical role of designing novel therapeutic strategies with antivirals.

Here, we have attempted to elaborate on the previous proposal for using a BSAA combination therapy for COVID-19. Our focus was a concept for further investigation, in other words utilizing already licensed BSAAs with some activity against SARS-CoV-2 concurrently with experimental RNA substrate mimetics as nsp15/NendoU inhibitors. To our knowledge, only in vitro evidence exists for such nuclease inhibitors to-date [ 36]. More lead optimization work on such RNA substrate mimetics to further establish proof of principle is now warranted at a minimum. These can include parallel cellular assays to characterize their stability within microsomes or nanoparticles and off-target properties. As such, the 'double-hit effect' is hypothesis-generating at present and additional supporting data would be necessary prior to advancement of this strategy into in vivo animal model studies and subsequent first-in-human clinical trials. Conversely, the CoV nsp14 would be an attractive target to combine with a BSAA; inhibition of the ExoN catalytic site can be expected to knock out this viral RNA proofreader, thus restoring the potency particularly of nucleoside analogs that are readily available antiviral agents [ 1, 18, 42, 47]. Unlike for NendoU, we are not yet aware of any reported inhibitor class specifically of the CoV exonuclease activity, which limits advancing this combination strategy for now.

We can only speculate on the challenges of RNA substrate mimetics vis-à-vis their translational potential. While the PK/PD profiles of long-standing nucleoside analogs are well established, the in vivo dynamics of potential RNA substrate mimetics are essentially unknown. Additionally, the optimal method or vehicle for their delivery remains to be determined. One possibility is the emerging RNA-based delivery system which can offer significant advantages in terms of simpler design and flexibility of manipulation in comparison to the more traditional viral subunit/vector-driven methods [ 49]. It is also noteworthy that mRNA is under investigation as a robust platform for at least one of the current leading vaccine candidates (mRNA-1273 by Moderna, Inc., MA, USA) which has rapidly been translated into Phase I clinical trial (ClinicalTrials.gov Identifier: NCT04283461). This involves a novel lipid nanoparticle-encapsulated mRNA formulation that encodes for the full-length S protein of SARS-CoV-2 which can be readily administered intramuscularly. In principle, given the relative ease of manufacturing RNA oligonucleotides compared with large recombinant viral proteins, we can anticipate quick adaptability of a similar delivery system to RNA substrate mimetics as cargo. Another important consideration is the temporality of administration of such antiviral therapy. As has been empirically noted, an initially dampened host innate immune response preceding a late exuberant cytokine storm are the hallmarks of severe COVID-19 immunopathology and associated mortality [ 50]. Given the current understanding of CoV-nsp15/NendoU as a major immunomodulator, counteracting viral evasion of host innate immunity in the preliminary stages of disease seems logical. Additionally, formation of double membrane vesicles and RTC activity dominate early during the coronavirus replication cycle before viral protein synthesis escalates to sufficient levels causing viremia and systemic infection. Therefore, the ideal time for inhibition of SARS-CoV-2 endonuclease activity within a combination strategy is perhaps quite early on in COVID-19.

Further, even if favorable PK/PD data are obtained through preclinical testing, the toxicity profiles of such therapeutics solo and in combination for drug-drug interactions would need to be rigorously evaluated in model in vivo systems. The 'double-hit hypothesis' may also dictate that SARS-CoV-2 proteases (i.e., PLpro and 3CLpro) could serve as the add-on third viral enzyme target on top of a backbone of RdRp inhibition with a nucleoside analog. On the contrary, 2′-O-Mtase activity, while possibly facilitating viral evasion of the host immune response through epigenetic RNA modification, may not be considered crucial for driving the replication cycle relative to other nsps of the RTC. Given no reported inhibitors of 2′-O-Mtase activity in SARS-CoV-2, we posit that this cannot be expected to rise to the significance of either hit 1 or 2 (see graphical abstract). Proteases also are not unique to the Nidovirales order per se; whether they can be of specific 'synthetic lethality' to coronaviridae when inhibited in combination remains to be seen.

Recent clinical trial data somewhat reinforces this doubt whether protease inhibition can be of significant benefit in the context of COVID-19. Lopinavir-ritonavir (protease inhibitor combination approved for HIV) did not produce any clinical benefit over standard of care in hospitalized adult patients with severe COVID-19 [ 51]. Upon reflection, this may not be surprising as lopinavir is designed to be an inhibitor of the retroviral protease which is different from the cysteine protease class of CoVs. While drug repurposing is a time-critical method, this experience should remind us of the fundamental principle of target specificity in preclinical development to enhance clinical success. Encouragingly, recent timely work has determined the crystal structure of SARS-CoV-2 main protease (Mpro) also known as 3CL protease (3CLpro). This in turn allowed the identification of potentially specific protease inhibitors with translational potential through a program of combined structure-based drug design, virtual and high-throughput drug screening [ 52, 53]. It must also be acknowledged that there is no historical precedent for RNA substrate mimetics in translational medicine, which renders them in uncharted waters. Alternatively, small molecule inhibitors of RNaseA have been proposed as a strategy to inhibit coronavirus nsp15 as it bears crystal structural similarity to RnaseA [ 33]. However, as the initial suboptimal efficacy of repurposed HIV protease inhibitors for COVID-19 has demonstrated to some degree, target specificity and substrate affinity must remain paramount even in this brave new world of a pandemic. While RNaseA inhibitors can shut down the endonuclease activity, off target effects in the host would be a concern that needs in vivo evaluation. Therefore, the current in vitro evidence, though limited on RNA substrate mimetics for their potential as specific CoV endonuclease inhibitors, can still serve as the basis for further confirmatory studies to validate the concept.

In conclusion, we hope this insight adds a new layer to a fast expanding armamentarium of therapeutic strategies against COVID-19. Nsp15/NendoU RNA substrate mimetics described in previous studies deserve to be revisited for preclinical optimization and testing, perhaps in combination with nucleoside analogs [ 36].

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

References

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46. Bao L, Deng W, Gao H. et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv (2020) (Epub ahead of print). [ Google Scholar]

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COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline.

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Abstract

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. 1., 2., 3. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5′ and 3′ of the uridine. 1., 2. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3′ of the recognition uridylate, with some cleavage 3′ of cytidylate. A highly conserved RNA structure in the 3′ non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn ²⁺. The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 Å structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.

Abbreviations used

SARS

severe acute respiratory syndrome

MALDI-TOF

matrix-assisted laser desorption/ionization time-of-flight

TLC

thin-layer chromatography

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RNA Recognition and Cleavage by the SARS Coronavirus Endoribonuclease

# data[].attributes.attachment_link.description
The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. 1., 2., 3. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study.

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Benzopurpurine B | C34H28N6NaO6S2+ | CID 54603169 - structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities, safety/hazards/toxicity information, supplier lists, and more.

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Encontrar el control positivo como posible inhibidor

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El paper1 cita a este autor con esas conclusiones

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Coronaviruses Encoding Mutant Forms of nsp15 Exhibit Impaired Replication in Macrophages.

Here we characterize two independent nsp15 mutant viruses of MHV-A59, termed N15m1 and N15m3, and report that these viruses exhibited impaired viral replication and induced rapid cell death during infection of mouse bone marrow-derived macrophages (BMDMs) ( Fig. 1). The parental WT MHV-A59 (termed MHV-WT) and all mutant viruses were generated using reverse genetics ( 24) and were subjected to deep sequencing to validate the genotypes. N15m3 contains an alanine substitution in the catalytic histidine (H262A) residue of nsp15, which was previously shown to inactivate endoribonuclease activity ( 17, 25). N15m1 contains a methionine substitution at a highly-conserved threonine residue (T98M) and an alanine substitution at arginine-971 (R971A) in nsp3 ( Fig. 1A and Fig. S1A). We also generated a mutant virus with a single substitution in nsp3 (R971A), termed N3m, to distinguish the effect of the substitution in nsp3 from the substitution in nsp15 of N15m1 virus. To date, we have been unable to isolate a virus containing only the T98M substitution, which may indicate that additional substitutions are necessary to stabilize the T98M mutant virus.

MHV-WT and the three mutant viruses replicate efficiently in a murine fibroblast cell line, 17Cl-1 cells, indicating that WT nsp15 is not required for viral replication in fibroblasts ( Fig. S1B), which is in agreement with a previous report describing nsp15 catalytic mutants ( 18). We report that infection of BMDMs generated from C57BL/6 mice (B6) with nsp15 mutant viruses resulted in limited production of progeny virus and more rapid cell death compared with WT virus infection ( Fig. 1 B and C and Fig. S1D). This result is in contrast to the robust replication of MHV-WT and N3m, which replicate to high titers in BMDMs with minimal cell death as late as 24 h postinfection (hpi) ( Fig. 1 B and C and Fig. S1D). Efficient replication of the nsp15 mutant viruses was restored in BMDMs generated from mice with a knockout of the type I IFN receptor ( ifnar−/−) ( Fig. 1D). In the absence of IFN signaling, all tested viruses induced similar cell death kinetics ( Fig. S1C and Fig. S1E). These results implicate type I IFN signaling as a major contributor to the replication deficiency of nsp15 mutants.

MHV nsp15 Mutant Viruses Stimulate an Early and Robust Induction of Type I IFN in Macrophages.

Given the importance of IFN signaling in controlling nsp15 mutant virus infection of macrophages, we hypothesize that WT nsp15 antagonizes the type I IFN system. To address this question, B6 BMDMs were infected with MHV-WT or mutant viruses and harvested at 8, 12, and 16 hpi to evaluate the level of IFN-αmRNA and protein. We found that the nsp15 mutant viruses stimulated an early and more robust induction of IFN-α compared with MHV-WT or N3m virus ( Fig. 2). IFN-α mRNA was significantly elevated at 8 hpi in nsp15 mutant-infected cells, with WT or N3m virus requiring 12 hpi to reach similar levels ( Fig. 2A). The detection of IFN-α mRNA in nsp15 mutant virus-infected cells peaked at 12 hpi and was slightly reduced at 16 hpi ( Fig. 2A), corresponding to a reduction in the number of viable cells ( Fig. 1C). We noted a significant reduction in the level of nucleocapsid (N) gene mRNA in nsp15 mutant virus-infected cells at 12 and 16 hpi ( Fig. 2B), time points when IFN-α expression was significantly elevated ( Fig. 2A). The concentration of IFN-α protein was significantly higher in cell supernatants obtained from nsp15 mutant-infected cells, compared with WT- or N3m-infected cells ( Fig. 2C). Additionally, increased detection of ISG54 in N15m1-infected cells was observed using immunofluorescence ( Fig. S2A). Consistent with a previous report ( 26), activation of IFN during MHV infection was dependent on production of MDA5 (encoded by the ifih1 gene), as BMDMs derived from ifih1−/− mice did not express IFN when infected by either WT or mutant viruses ( Fig. 2D and Fig. S2B). Overall, these results demonstrate that nsp15 mutant viruses stimulate an early and robust type I IFN response in B6 BMDMs, indicating that nsp15 functions as a type I IFN antagonist during MHV infection.

MHV nsp15 Mutant Viruses Trigger Rapid Apoptotic Cell Death in Macrophages.

Because we observed rapid cell death in nsp15 mutant-infected macrophages ( Fig. 1C and Fig. S1D), we investigated the cell death pathways triggered during viral replication. We first evaluated the effects of various inhibitors on virus-infected cells. The pan-caspase inhibitor zVAD ( 27) prevented virus-induced cell death ( Fig. 3A). In contrast, neither Necrostatin-1 (Nec-1), a receptor interacting serine/threonine kinase 1 (RIPK1) inhibitor ( 28), nor caspase-1 inhibitor VX-765 ( 29), prevented virus-induced cell death ( Fig. 3A). These data suggest that infection of BMDMs by nsp15 mutant viruses activates apoptotic cell death rather than RIPK1/RIPK3-dependent necroptosis or caspase-1-mediated pyroptosis ( 30, 31). However, we note that zVAD-a cysteine protease inhibitor-may also affect viral replication ( 32); therefore, we investigated other hallmarks of apoptotic cell death, including the activation of caspase-3/7. We observed enhanced caspase-3/7 activity in nsp15 mutant virus-infected BMDMs ( Fig. 3B). Furthermore, increased levels of cleaved caspase-3 products were detected by Western blotting ( Fig. 3C). Finally, condensed chromatin and nuclear fragmentation were observed by electron microscopy in nsp15 mutant-infected cells, but not in BMDMs infected by MHV-WT ( Fig. S3A). Taken together, these data demonstrate that nsp15 mutant viruses trigger early apoptosis in macrophages.

To evaluate the possibility that the observed apoptotic cell death was induced by IFN-α production alone, we treated B6 BMDMs with various doses of IFN-α and evaluated caspase-3/7 activity. We found that IFN-α treatment did not induce caspase-3/7 activity ( Fig. S3B). Furthermore, no elevated caspase-3/7 activity was observed in BMDMs treated with UV-inactivated supernatant, which contained replication-defective virus and IFN-α ( Fig. S3B). These results indicate that active virus replication, and not IFN-α alone, is required for induction of apoptosis. Using a complementary approach, we evaluated virus-induced apoptosis in ifih1−/− BMDMs. These cells lack MDA5 expression and, as shown in Fig. 2D, do not induce IFN during viral infection. However, other dsRNA-sensing pathways remain intact in these cells. We found that infection of ifih1−/− BMDMs with nsp15 mutant viruses induced elevated levels of caspase-3/7, which promoted rapid cell death ( Fig. 3 D and E). These results implicate a non-MDA5-dependent pathway as an additional mechanism in the induction of apoptosis. Taken together, these data indicate that nsp15 mutant virus infection can activate multiple independent dsRNA-sensing pathways to induce apoptotic cell death.

MHV nsp15 Mutant Viruses Activate Host dsRNA Sensors.

Because our data suggest that additional dsRNA-sensing pathways are activated in response to nsp15 mutant virus replication, we investigated other dsRNA-sensing pathways that have been linked to apoptotic cell death. To this end, we examined the protein kinase R (PKR) and the 2′-5′ oligoadenylate synthetase (OAS)/RNase L systems ( 33⇓ ⇓ ⇓ - 37). First, we evaluated the level of phosphorylated eIF2α, an indicator of PKR activation ( 35, 37), during infection. Increased phosphorylation of eIF2α was observed in BMDMs infected with nsp15 mutant viruses compared with infection with WT virus ( Fig. 4A). Addition of C16, a specific kinase inhibitor of PKR ( 38), significantly reduced the levels of caspase-3/7 activity ( Fig. 4B) and prevented cell death ( Fig. S4A). These data suggest that the PKR pathway is activated during MHV infection of BMDMs. We also assessed the degradation of RNA, which is an indicator of active 2′-5′ OAS/RNase L system signaling ( 39). Degradation of RNA was observed as early as 8 hpi in cells infected with the nsp15 mutant viruses, but not in cells infected by MHV-WT or N3m ( Fig. 4C and Fig. S4B). Degradation of RNA was not inhibited by blocking apoptosis with either zVAD or C16 ( Fig. 4D). This result indicated that activation of OAS/RNase L was independent of PKR activation. In addition, robust RNA degradation was observed in nsp15 mutant virus-infected ifih1−/− BMDMs ( Fig. S4C), suggesting that the OAS/RNase L system was activated in the absence of IFN. Finally, neither RNA degradation ( Fig. S4D) nor elevated caspase-3/7 activity ( Fig. S4E) was observed in ifnar−/− BMDMs despite the fact that robust viral replication was present ( Fig. 1D). This could be because of low basal expression of PKR ( Fig. S4F) and OAS in ifnar−/− macrophages, the latter of which was previously reported ( 40). Taken together, these data indicate that nsp15 mutant viruses activate multiple independent host dsRNA-sensing systems in macrophages, including MDA5, PKR, and OAS/RNase L. In the absence of functional nsp15, the collective stimulation of these pathways by viral dsRNA results in robust type I IFN induction, apoptotic cell death, and severely reduced viral titer.

T98M Mutation Destabilizes nsp15 Protein and Impairs Endoribonuclease Activity.

As shown above, the N15m1 and N15m3 viruses repeatedly exhibited similar phenotypes, despite encoding different mutations in nsp15. Because the N15m1 mutant virus is unique to this study, we investigated the effect of the T98M mutation on the nsp15 protein. The T98M mutation resides at the interface between the N-terminal domain and middle domain of nsp15 ( Fig. 1A, Right). The endoribonuclease activity of nsp15 requires the formation of stable oligomers, which may be destabilized by the T98M mutation ( 25). Western blot analysis revealed that levels of nsp15 were significantly reduced in both N15m1- and N15m3-infected cells compared with WT-infected cells ( Fig. 5A, Left). It is possible that this phenotype could be because of the limited viral replication of nsp15 mutant viruses observed in B6 BMDMs or reduced detection of nsp15 mutant proteins by antiserum. However, compared with N15m3, the level of N15m1 nsp15 protein remained low during infection of ifnar−/− BMDMs, despite similar N protein levels ( Fig. 5A, Right) and growth kinetics for all viruses tested ( Fig. 1D). We also note that detection of nsp15 protein by antiserum was not affected by the T98M mutation, as both recombinant WT and T98M nsp15 were equally detectable ( Fig. 5B, Lower). These results suggest that the T98M mutation destabilizes nsp15, resulting in a decrease in the steady-state level of the protein in N15m1-infected cells.

To further evaluate the effect of the T98M mutation on the nsp15 protein, we cloned and expressed codon-optimized versions of the WT and T98M nsp15 as SUMO-fusion proteins in Escherichia coli ( Fig. 5B, Upper). Based on the structure of nsp15, the SUMO tag will not affect the assembly of oligomers, including hexamers, as previously reported ( 12⇓ - 14). To determine if the T98M mutation destabilizes the protein, differential scanning fluorimetry (DSF) was used to measure the stability of nsp15 in response to heat ( 41). DSF assesses the denaturation of proteins in the presence of a hydrophobic dye that will fluoresce upon binding to hydrophobic protein sequences. This technique has been used to monitor interactions between viral protein subunits ( 42). WT nsp15 undergoes a major transition to the denatured state at 47 °C ( Fig. 5C). In contrast, the T98M mutant denatured at 40 °C, 7 °C lower than the WT nsp15, which is consistent with our hypothesis that the T98M mutation renders nsp15 less stable. Using dynamic light-scatter spectrometry, we report that the majority of WT nsp15 assembles to form oligomers at a protein concentration of 0.05 mg/mL. In contrast, the majority of the T98M nsp15 protein is detected in the monomeric form under these conditions, suggesting that the T98M mutation impairs nsp15 oligomerization ( Fig. S5). Thus, our in vitro characterization data for WT and T98M nsp15 proteins strongly support our prediction that the T98M mutation decreases protein stability. We reasoned that the instability of nsp15 imposed by the T98M mutation might impact endoribonuclease activity, which may in turn contribute to the N15m1 phenotype during infection. Indeed, the T98M mutant exhibited significantly reduced RNA cleavage activity in vitro compared with WT protein ( Fig. 5D). Altogether, these results demonstrate that the T98M mutation, like H262A, impairs nsp15 endoribonuclease function, albeit by different means.

Loss of nsp15 Endoribonuclease Activity Affects the Distribution of dsRNA in Virus-Infected BMDMs.

Based on our findings that highlight the importance of nsp15 endoribonuclease activity, we initially hypothesized that nsp15 may prevent accumulation of viral dsRNA in the cytoplasm, thereby reducing the likelihood of triggering host dsRNA sensors. To address this possibility, we measured the abundance of dsRNA during infection of BMDMs by flow cytometry. We predicted that the loss of nsp15 endoribonuclease activity in the mutant viruses would correspond to an increase in immunofluorescence intensity of dsRNA. However, we did not observe a significant difference between WT- and N15m3-infected cells in either B6 or ifnar−/− BMDMs at early or late phases of infection ( Fig. S6 A-C), implying that nsp15 does not reduce the amount of dsRNA in infected cells.

Previous studies reported that coronavirus dsRNA associates with replication complexes early during infection and is buried in DMVs, which are thought to protect viral RNA from host sensors ( 8, 9, 43). Additionally, our group and others have shown that nsp15 also associates with replication complexes ( 44, 45) and interacts with single-strand RNA and dsRNA ( 13). Therefore, we hypothesized that that nsp15 may function to maintain the association of dsRNA with replication complexes. We predicted that loss of nsp15 endoribonuclease activity may allow for accumulation of dsRNA that is not associated with replication complexes ("free" dsRNA). Such free dsRNA might be vulnerable to detection by cytoplasmic dsRNA sensors. To test this hypothesis, we used ifnar−/− BMDMs because we showed that replication of both WT and nsp15 mutant viruses is equivalent in these cells. The subcellular localization of dsRNA and nsp2/3, which are hallmark components of viral replication complexes ( 8), was detected using confocal microscopy with specific antibodies. Interestingly, N15m3 infection yielded more dsRNA foci that did not colocalize with nsp2/3 foci ( Fig. 6A). To quantify these free dsRNA foci, we used an automated immunofluorescence analysis program (IMARIS) to identify and count the number of dsRNA and nsp2/3 foci. We found that there were similar levels of nsp2/3⁺ replication complexes in N15m3- and WT-infected cells at 6 hpi; however, more free-dsRNA foci were detected in N15m3-infected cells than in WT-infected cells ( Fig. 6A). Consistent with previous reports ( 43, 46), dsRNA was dispersed late in infection, leading to a similar increase in the number of free-dsRNA foci in both WT- and N15m3-infected cells ( Fig. S6D). We note that the early dispersal pattern of dsRNA in N15m3-infected cells coincides with the early activation of dsRNA sensors and the IFN response ( Figs. 2- 4). Taken together, these data suggest that nsp15 may not affect the abundance of cytosolic dsRNA, but rather functions to maintain the association of dsRNA with replication complexes, likely by sequestering dsRNA within DMVs ( 8).

To further elucidate the relationship between nsp15 and dsRNA during viral infection, we visualized nsp15 and dsRNA localization by immunofluorescence using specific antibodies. As expected, we found that the number of dsRNA foci that colocalized with nsp15 foci was significantly reduced in N15m3-infected cells compared with WT-infected cells ( Fig. 6B). These results demonstrate that nsp15 localization with dsRNA-and therefore with replication complexes-is dependent on endoribonuclease activity, the lack of which may result in more free cytoplasmic dsRNA available for detection by host sensors.

nsp15 Mutations Profoundly Attenuate Murine Coronavirus Infection in Vivo.

Because nsp15 mutant viruses induce a robust IFN response and activate host dsRNA sensors, we wanted to determine if the loss of nsp15-mediated antagonism of innate immune responses alters the pathogenesis of murine coronavirus. We first infected C57BL/6 mice intraperitoneally with 6 × 10 ⁴ plaque-forming units (pfu) of WT or mutant virus and evaluated viral burden in target organs and pathology in the liver. Strikingly, standard plaque assays that enumerate infectious particles failed to detect virus in the livers and spleens of mice inoculated with nsp15 mutant viruses at 3 and 5 d postinfection (dpi) ( Fig. 7A). However, high viral titers were detected in the same target organs of mice infected with WT or N3m virus ( Fig. 7A and Fig. S7A). N gene transcripts, albeit low levels, were only detectable by qPCR in the mesenteric lymph node (MLN) of mice infected with nsp15 mutant virus at 1 dpi ( Fig. 7B). Histological examination of the livers revealed typical lesions associated with infection by MHV-WT and N3m, but not in nsp15 mutant virus-infected mice ( Fig. 7C and Fig. S7B).

A lethal challenge model of MHV-A59 was used to determine if nsp15 mutant viruses are attenuated. Injection of 600 pfu of WT virus into the mouse cranium is sufficient to induce lethal encephalitis ( 47, 48). As expected, all WT- or N3m-infected mice lost body weight ( Fig. 7D and Fig. S7C) and succumbed to infection by 7-9 dpi ( Fig. 7E and Fig. S7D). In contrast, all nsp15 mutant virus-infected mice survived the infection ( Fig. 7E) and exhibited only mild or transient weight loss, from which they recovered over time ( Fig. 7D). These data reveal that nsp15 mutant viruses are profoundly attenuated and exhibit minimal pathogenicity, even in this sensitive model. To evaluate the replication of these viruses in the absence of IFN signaling, ifnar−/− mice were intraperitoneally infected with 50 pfu of WT or nsp15 mutant virus. All mice succumbed to infection, indicating that all tested viruses could replicate and induce lethal disease in the absence of IFN signaling ( Fig. 7F). Interestingly, N15m1-infected mice exhibited delayed mortality compared with WT-infected mice ( P = 0.0047), suggesting that either the instability of nsp15 or the addition of the mutation in nsp3 may contribute to viral attenuation.

MHV nsp15 Mutant Viruses Confer Protective Immunity against WT Virus Infection.

Because the nsp15 mutant viruses are highly attenuated for virulence in vivo, we wanted to determine if these viruses could elicit protective immunity against subsequent challenge with WT virus. B6 mice were infected intraperitoneally with 6 × 10 ⁴ pfu of nsp15 mutant viruses. Four weeks later, the same mice, as well as naïve, age-matched mice, were challenged with 6 × 10 ⁴ pfu of WT virus and viral burden and liver pathology were assessed at 5 dpi. In contrast to naïve mice, mice that had been inoculated with nsp15 mutant viruses before challenge with MHV-WT produced undetectable viral titers in tested organs ( Fig. 8A) and no liver pathology observed ( Fig. 8B). Even more striking were the results from intracranial infection. Here we challenged mice that had been previously inoculated 7 wk prior with N15m1 or naïve mice with a 10-fold lethal dose of MHV-WT. We found that the immunized mice experienced only minor weight loss and fully recovered from infection, whereas the age-matched naïve mice succumbed to infection ( Fig. 8 C and D). This finding demonstrates that immunization with nsp15 mutant virus protects mice from a subsequent lethal challenge, suggesting that nsp15 mutant viruses can elicit strong, protective immune memory in mice, which highlights their potential as vaccine candidates.

Cells, Viruses, Antibodies, and Chemicals.

Baby hamster kidney cells expressing the MHV receptor (BHK-R) were kindly provided by Mark Denison, Vanderbilt University Medical Center, Nashville, TN. The murine fibroblast 17Cl-1 cell line was maintained in DMEM containing 5% FCS DMEM. The L929 cell line was a gift of Francis Alonzo, Loyola University Chicago, Maywood, IL. Differentiated BMDMs were maintained in bone marrow macrophage media containing DMEM (#10-017-CV, Corning) supplemented with 30% L929 cell supernatant, 20% FCS, 1% l-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin. WT MHV strain A59 (GenBank accession no. AY910861) was generated by reverse genetics and full-genome-sequenced. Rabbit anti-nsp2/3 serum (anti-D3) and anti-nsp15 serum (anti-D23) were previously reported by our laboratory ( 8, 44). Mouse anti-N (J3.3) was a gift from John Fleming (University of Wisconsin-Madison, WI). The following antibodies were purchased commercially (catalog number and company indicated): mouse anti-β-actin (#A00702, Genscript), donkey anti-rabbit-HRP (#711-035-152, Jackson ImmunoResearch), goat anti-mouse-HRP (#1010-05, SouthernBiotech), anticleaved-caspase-3 (#Asp175, Cell Signaling Technology), anti-dsRNA (#K1, Scicons), anti-ISG54 (#PA3845, ThermoFisher), anti-eIF2α (#sc-133132, Santa Cruz), and anti-p-eIF2α (#sc-12412, Santa Cruz). Chemical inhibitors were obtained from the following sources: pan-caspase inhibitor zVAD (#627610, Millipore), Nec-1 (#480065, Millipore), PKR inhibitor C16 (#527450, Millipore), and VX-765 (#F7120, UBPbio), staurosporine (#ALX-380-014, Enzo Life Sciences).

Mutant Viruses and Deep Sequencing.

To generate MHV mutant viruses, nucleotide changes were incorporated into the MHV-A59 genome through PCR mutagenesis (primers available upon request) of cDNA fragments. Subsequent generation of virus by reverse genetics was performed as previously described by Yount et al. ( 24). Viral genomic RNA from in vitro transcription (mMESSAGE mMACHINE T7 Transcription Kit; AM1344, Invitrogen Ambion) of ligated cDNA fragments was electroporated into BHK-R cells. Cell supernatant was collected as viral stock following observation of cytopathic effects. Infectious clones were plaque-purified, propagated on BHK-R cells, and titrated on 17Cl-1 cells. Mutant viruses were maintained exclusively in BHK-R cells, which do not produce or respond to IFN. All virus stock preparations and plaque-purified isolates used in this study were full-genome deep-sequenced (Kansas State University diagnostic laboratory). Briefly, viral RNA was extracted from virus stock using QIAamp MinElute Virus Spin Kit (57704, Qiagen), used to generate a cDNA Library and sequenced by Miseq or Ion Torrent technology. Mutant MHV sequences were aligned to the WT MHV-A59 synthetic construct (GenBank accession no. AY910861).

Infection and Mouse Experiments.

BMDMs in 12- or 24-well plates were infected with indicated viral strains at a multiplicity of infection (MOI) of 0.1 or 1 in serum-free media. For growth kinetics analysis, cell-culture supernatants were collected at indicated time points and titrated by plaque assay on 17Cl-1 cells. For mouse infection, all experiments were performed using protocols reviewed and approved by the Loyola University Chicago Institutional Animal Care and Use Committee (IACUC). For intracranial infections, 6-wk-old C57BL/6 female mice (Jackson Laboratory) were inoculated with 600 pfu virus and monitored for body weight daily and killed when weight loss was over 25% according to the IACUC protocol. For intraperitoneal infection, 6-wk-old mice were injected with 60,000 pfu and organs were collected at indicated time points. Evidence of liver pathology was determined by H&E staining.

Cell Death Assays.

Cell viability and caspase-3/7 activity were measured using CellTiter Glo (G7571, Promega) and Caspase-Glo 3/7 (G8091, Promega), respectively, according to the manufacturer's protocols.

DSF Assay.

Recombinant MHV nsp15 or T98M proteins were diluted in storage buffer (10% glycerol, 20 mM Tris⋅Cl, pH 7.5, 300 mM NaCl, and 5 mM β-ME) at different concentrations. Size measurement was carried out by Zetasizer Nano-S dynamic light scattering (Malvern Instruments) at room temperature. Each sample was measured at least three times. The average intensity and size distributions are shown in Fig. S5.

RNA Cleavage Assay.

The standard RNA cleavage assay used 1 × 10 ⁴ cpm of 5′-end radiolabeled RNA substrate (1 μM final RNA concentration) and 0.026 μM nsp15 in 50 mM Tris⋅HCl (pH 7.5), 50 mM KCl, 1 mM DTT, and 5 mM MnCl ₂ at 30 °C. The endoribonuclease reactions were terminated by the addition of a gel-loading buffer that contained 7.5 M urea. Products were separated by electrophoresis in 20% polyacrylamide gels containing 7.5 M urea. Gels were wrapped in plastic and exposed to a PhosphorImager screen for quantification using Molecular Dynamics software.

Cells, Viruses, Antibodies, and Other Reagents.

Baby hamster kidney cells expressing the MHV receptor (BHK-R) were kindly provided by Mark Denison, Vanderbilt University Medical Center, Nashville, TN, and cultured in DMEM (#12100-046, Gibco) supplemented with 10% heat-inactivated FCS and G418 (0.8 mg/mL) (SV30069, HyClone) to maintain selection for MHV receptor expression. The 17Cl-1 cell line was maintained in DMEM containing 5% FCS DMEM. The L929 cell line was a gift of Francis Alonzo, Loyola University Chicago, Maywood, IL, and maintained in DMEM (#10-017-CV, Corning) supplemented with 10% FCS, 1% l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin. Differentiated BMDMs were maintained in bone marrow macrophage (BMM) media containing DMEM (#10-017-CV, Corning) supplemented with 30% L929 cell supernatant, 20% FCS, 1% l-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin. WT MHV strain A59 (GenBank accession no. AY910861) was generated by reverse genetics and full-genome-sequenced. Rabbit anti-nsp2/3 serum (anti-D3) and anti-nsp15 serum (anti-D23) were previously reported by our laboratory ( 8, 44). Mouse anti-N (J3.3) was a gift from John Fleming, University of Wisconsin-Madison, WI. The following antibodies were purchased commercially (catalog number and company indicated): mouse anti-β-actin (#A00702, Genscript), donkey anti-rabbit-HRP (#711-035-152, Jackson ImmunoResearch), goat anti-mouse-HRP (#1010-05, SouthernBiotech), anticleaved-caspase-3 (#Asp175; Cell Signaling Technology), anti-dsRNA (#K1, Scicons), anti-ISG54 (#PA3845, ThermoFisher), anti-eIF2α (#sc-133132, Santa Cruz), and anti-p-eIF2α (#sc-12412, Santa Cruz). Chemical inhibitors were obtained from the following sources: pan-caspase inhibitor zVAD (#627610, Millipore), Nec-1 (#480065, Millipore), PKR inhibitor C16 (#527450, Millipore), VX-765 (#F7120, UBPbio), and staurosporine (#ALX-380-014, Enzo Life Sciences).

Generation of BMDM.

Bone marrow was collected from femurs of C57BL/6 (#000664, The Jackson Laboratory), ifih−/− (#015812, The Jackson Laboratory), or ifnar−/− mice (breeder pair provided by Deborah Lenschow, Washington University in St. Louis, St. Louis, MO). For generation, 5 × 10 ⁶ bone marrow cells were plated in 100 × 26-mm Petri dishes (25387-030, VWR) with 15 mL BMM media with 50 µM β-mercaptoethanol. After 3 d of incubation at 37 °C with 5% CO ₂, 10 mL of BMM media was added. Following another 3 d of differentiation, BMDMs were washed in cold 1× PBS, incubated for 30 min at 4 °C in 10 mL 1×, PBS then gently rinsed from the plates by manual pipetting. Next, 1 × 10 ⁷ cells/mL were suspended in BMM media with 10% DMSO and stored in liquid nitrogen until use. For preparation of L929 cell supernatant, 3.75 × 10 ⁵ L929 cells were plated in 75 mL DMEM in a T150 flask (#10-126-34, Thermofisher). After 6 d of incubation at 37 °C/5% CO ₂, the supernatant was harvested, filtered, and stored at −20 °C until use. For viral infections, BMDMs were thawed and plated in 100 × 26-mm Petri dishes in BMM media without β-mercaptoethanol. After 3 d of incubation at 37 °C/5% CO ₂, cells were plated onto tissue culture dishes for subsequent infection experiments after 24-h incubation.

Mutant Viruses and Deep Sequencing.

To generate MHV mutant viruses, nucleotide changes were incorporated into the MHV-A59 genome through PCR mutagenesis (primers available upon request) of cDNA fragments. Subsequent generation of virus by reverse genetics was performed as previously described by Yount et al. ( 24). Viral genomic RNA from in vitro transcription (mMESSAGE mMACHINE T7 Transcription Kit; AM1344, Invitrogen Ambion) of ligated cDNA fragments was electroporated into BHK-R cells. Cell supernatant was collected as viral stock following observation of cytopathic effects. Infectious clones were plaque-purified, propagated on BHK-R cells, and titrated on 17Cl-1 cells. Mutant viruses were maintained exclusively in BHK-R cells, which do not produce or respond to IFN. All virus stock preparations and plaque-purified isolates used in this study were full-genome deep-sequenced (Kansas State University diagnostic laboratory). Briefly, viral RNA was extracted from virus stock using QIAamp MinElute Virus Spin Kit (57704, Qiagen), used to generate a cDNA Library and sequenced by Miseq or Ion Torrent technology. Mutant MHV sequences were aligned to the WT MHV-A59 synthetic construct (GenBank accession no. AY910861).

Viral Titration by Plaque Assay.

To determine virus growth kinetics, BMDMs in 24-wells plates were infected with indicated viral strains at an MOI of 0.1 in serum-free media. After 1-h incubation, inoculum was replaced with fresh, complete medium. Cell culture supernatants were collected at indicated time points and titrated by plaque assay on 17Cl-1 cells. To determine the viral titer in organs, a portion of tissue was homogenized with 1.0-mm diameter zirconia/silicon beads (11079110z, BioSpec Products) using an automated homogenizer (MP Biomedicals) at 6.0 m/s for 40 s for 50-mg tissue in serum-free DMEM. The homogenized organs were centrifuged at 450 × g for 5 min and the supernatants were titrated for viral plaques on 17Cl-1 cells. Titers were obtained from three independent assays for each sample. Graphs of virus kinetics were generated using Prism software (GraphPad Software).

Cell Death Assays.

Cell viability and caspase-3/7 activity were measured using CellTiter Glo (G7571, Promega) or Caspase-Glo 3/7 (G8091, Promega), respectively, according to the manufacturer's protocol. Briefly, 3.0 × 10 ⁵ BMDMs per well were plated in 24-well plates and infected at an MOI of 0.1 for 1 h in serum-free media. Media was replaced with complete media, or for addition of chemical inhibitors or IFN-α, viral inoculum was replaced with completed BMM media containing indicated concentration of inhibitors or IFN-α. Infected BMDMs were incubated for 24 h at 37 °C/5% CO 2. BMDMs were washed twice with 1× PBS and lysed with 150 µL Glo reagent/medium mixture (1:1); 50 µL of cell lysate was used for measuring the luminescence signal.

Immunofluorescence.

BMDMs (1.5 × 10 ⁵ cells per well) were plated on a glass coverslip in a 24-well plate for 24 h and subsequently infected with WT or mutant virus at an MOI of 0.1 in serum-free media. At 6 or 12 hpi, the infected cells were fixed with 4% formaldehyde in 0.095 M Pipes buffer (P1851, Sigma), permeabilized with 0.1% Triton X-100 (T8787, Sigma) in 1× PBS, and blocked with 5% NGS. Primary and secondary antibodies were used as follows: anti-NSP2/3 (1:1,500), anti-dsRNA (1:500), anti-ISG54 (1:500), anti-nucleocapsid (1:500), donkey anti-rabbit IgG alexafluor 488 (1:1,000; A-21441, Invitrogen), and goat anti-mouse IgG Alexa Fluor 568 (1:1,000; A11004, Thermofisher). Nuclei were visualized with Hoescht 33342 (1:2,500; H1339, Life Technologies). Cells were imaged by collecting z-stack images with a Deltavision wide-field fluorescent microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ, Photometrics). Images were taken with a 20× or 100× lens. Samples were excited with light generated by an Insight SSI solid-state illumination module (Applied Precision, GE) and deconvolved with SoftWoRx deconvolution software (Applied Precision, GE). All images were collected under identical acquisition conditions and processed using Imaris 7.6.4 (Bitplane).

FACS Analysis.

Triplicate wells of BMDMs (6.0 × 10 ⁵) were plated in a 12-well plate (Corning) for 6 hpi experiments or (3.0 × 10 ⁵) in a six-well plate (Corning) for 12 hpi experiments to lessen syncytia formation. After 24 h, BMDMs were subsequently infected with WT or mutant virus at an MOI of 0.1. At 6 or 12 hpi, cells were collected and fixed with 4% formaldehyde in 1× PBS, permeabilized with 0.1% Triton X-100 in 1× PBS, and blocked with 1× PBS containing 5% NGS and 0.5% sodium azide. Cells were labeled with fixable viability dye (65-0865-14, eBioscience), anti-dsRNA (5 µg/mL), and goat anti-mouse (1:1,000) secondary antibody labeled with Alexa Fluor 568. Cells were analyzed using a LSR Fortessa cell analyzer (BD Bioscience). Flow cytometry data were analyzed using FlowJo software (TreeStar).

Quantification of IFN-α Production by RT-qPCR and ELISA.

BMDMs in a 12-well plate were mock-infected or infected with virus at an MOI of 0.1. At indicated time points, monolayer cells were used for RNA extraction using an RNeasy Mini Kit (74104, Qiagen), and cell culture supernatants were collected for ELISA. To determine IFN-α11, β-actin, or MHV-A59 N gene mRNA production, total RNAs were extracted and an equal amount of RNA (500 ng) was used for cDNA synthesis using Rt ² HT First Strand Kit (330401, Qiagen). qPCR was performed with specific primers for mouse IFN-α11 (PPM03050B-200, Qiagen), mouse β-actin (PPM02945B-200, Qiagen) or MHV-A59 N gene using RT ² SYBR Green qPCR Mastermix (330502, Qiagen) in the Bio-Rad CFX96 system. The thermocycler was set as follows: one step at 95 °C (10 min), 40 cycles of 95 °C (15 s), 60 °C (1 min) and plate read, one step at 95 °C (10 s), and a melt curve from 65 °C to 95 °C at increments of 0.5 °C/0.05 s. Samples were evaluated in triplicate and data are representative of three independent experiments. The levels of mRNA were relative to β-actin mRNA and expressed as 2−ΔCT [ ΔC T = C T(gene of interest) − C T(β-actin)]. To measure secreted IFN-α, 50 µL cell culture supernatant was used for assay using a mouse IFN-α ELISA kit (BMS6027, eBioscience) following the manufacturer's instructions.

Electron Microscopy.

B6 BMDMs (6.0 × 10 ⁵ per well) in a 12-well plate were infected with virus at an MOI of 0.1 in serum-free DMEM. Control cells were treated with BMM media. Following a 1-h incubation at 37 °C/5% CO ₂, media was replaced with BMM media. Uninfected controls were treated with 1 µM staurosporine prepared in BMM media to induce apoptosis. Cells were further incubated at 37 °C/5% CO ₂ for 16 h then prepared for EM. Cells were washed in 1× PBS then incubated for 30 min at 4 °C in 1× PBS. Cells were gently collected from plates using a pipette, pelleted at 1,200 rpm for 5 min at 4 °C, and fixed (4% glutaraldehyde in 0.1 M cacodylate buffer). Cell sections were imaged by the Electron Microscopy Core Facility at Loyola University Chicago.

Western Blotting.

B6 BMDMs (12.0 × 10 ⁵ per well) were media-treated or infected with virus at an MOI of 0.1 in serum-free DMEM for 1 h at 37 °C/5% CO ₂. At indicated time postinfection, cells were lysed with cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium ortho-vanadate, 1 µg/mL leupeptin, 1 mM PSMF). Cells in lysis buffer were incubated on ice for 10 min then pelleted at 14,000 rpm for 10 min at 4 °C. Cell-free supernatant was diluted in 2× sample buffer (10% glycerol, 5% β-ME, 3% SDS, 7.5 mg/mL Trizma-base, bromophenol blue). Samples were separated by electrophoresis through 10% or 4∼15% gradient acrylamide PAGE-gel and transferred to PVDF Immuno-Blot membranes (162-0177, Bio-Rad). Membranes were suspended in blocking buffer (5% BSA in TBST) for 2 h at room temperature. Primary and secondary antibodies were used as follows: anticleaved-caspase-3 (1:1,000), anti-β-actin (1:5,000), anti-N protein (1:200), anti-p-eIF2α (1:600), anti-eIF2α (1:600), donkey anti-rabbit-HRP (1:5,000), goat anti-mouse-HRP (1:5,000), mouse anti-goat-HRP (1:5,000). Membranes were washed three times with 1× TBST and chemiluminescence was visualized using Western Lightning Plus-ECL reagent (50-904-9326, PerkinElmer) in a FluorChem E machine (ProteinSimple).

Purification of the Recombinant MHV nsp15 Protein from Escherichia coli.

The cDNA sequences of WT MHV nsp15 and T98M were amplified using the appropriate primers and subcloned into the pET-His-SUMO vector using fusion PCR. The constructs harboring the WT nsp15 or T98M mutant gene were transformed into Rosetta (DE3) pLys cells. A single colony was inoculated in 100 mL of Terrific Broth (TB) in the presence of 50 μg/mL of ampicillin and 17 μg/mL chloramphenicol and incubated at 37 °C overnight. The culture was then transferred into 3 L of TB media and cultured until the OD ₆₀₀ reached 0.8. The temperature was decreased to 16 °C and isfopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM for 20 h. The cells were collected by centrifugation at 8,000 × g for 10 min and cell pellets were suspended in lysis buffer containing 10% glycerol, 50 mM Hepes (pH 7.0), 400 mM NaCl, 5 mM β-ME, and 10 mM imidazole. The lysate was centrifuged at 15,000 × g for 30 min and the supernatant was loaded onto a Ni-NTA column. The Ni-NTA column was subsequently washed three times with lysis buffer containing 20 mM imidazole. The protein was eluted with elution buffer containing 500 mM imidazole and then the buffer was changed to 10% glycerol, 20 mM Tris⋅Cl (pH 7.5), and 5 mM β-ME. The sample was further purified by Mono Q-Sepharose with a gradient of Tris buffer containing 0-1 M NaCl. nsp15 was quantified by SDS/PAGE compared with a known concentration of BSA, and stored at −80 °C in a buffer containing 10% glycerol, 20 mM Tris-Cl, pH 7.5, 300 mM NaCl, and 10 mM β-ME.

Dynamic Light Scattering.

Recombinant MHV nsp15 or T98M proteins were diluted in storage buffer (10% glycerol, 20 mM Tris-Cl, pH 7.5, 300 mM NaCl, and 5 mM β-ME) at different concentrations. Size measurement was carried out by Zetasizer Nano-S dynamic light scattering (Malvern Instruments) at room temperature. Each sample was measured at least three times. The average intensity and size distributions are shown in Fig. S5.

DSF Assay.

DSF was carried out in the Stratagene MX3005P real-time PCR machine. The samples contain 1× SYPRO Orange, 10 μM of the recombinant protein. All samples were heated at a rate of 0.5 °C/min, and the fluorescence intensity and Tm (melting temperature) were determined.

Bioanalyzer RNA Analysis.

Equal amounts of total RNA purified from BMDMs were analyzed on an Agilent 2100 Bioanalyzer using RNA Nano LabChips.

Mouse Experiments.

All experiments were performed using protocols reviewed and approved by the Loyola University Chicago IACUC. C57BL/6 mice were purchased from the Jackson Laboratory. For intracranial infections, 6-wk-old mice were inoculated with 600 pfu in 20 µL PBS. Infected mice were monitored for body weight daily and euthanized when weight loss was over 25% according to the IACUC protocol. Statistical analysis of survival rate was evaluated using the log-rank test. For intraperitoneal infection, 6-wk-old mice were injected with 60,000 pfu in 100 µL PBS. Organs were collected at indicated time points and evaluated for viral replication. Liver pathology was evaluated using H&E staining by the Tissue Processing Core Facility at Loyola University Chicago.

Sequence Alignment.

Nsp15 T98 region from representative strains of coronavirus subgroups using Clustal W. α-Coronavirus: NL63 (Amsterdam I strain, AY567487), PEDV (CV777 strain, NC_003436); β-coronavirus: MHV, (A59 strain, AY910861); SARS-CoV (MA15 strain, FJ882957); MERS-CoV (EMC strain, JX869059); γ-coronavirus: IBV (Beaudette strain, NC_001451); δ-coronavirus: PDCoV (KJ567050).

View Abstract

# data[].attributes.attachment_link.title
Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

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Macrophages are immune cells equipped with multiple double-stranded RNA (dsRNA) sensors designed to detect viral infection and amplify innate antiviral immunity. However, many coronaviruses can infect and propagate in macrophages without activating dsRNA sensors. Here we present a function of murine coronavirus nonstructural protein 15 in preventing detection of viral dsRNA by host sensors.

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ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 - a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.

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Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2

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Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals.

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Si bien inicialmente se pensó que Nsp15 participaba directamente en la replicación viral, más tarde se demostró que los coronavirus deficientes en Nsp15 eran viables y se replicaban, lo que aumentó las dudas sobre el papel de la enzima en ese proceso. Más recientemente, se propuso que la actividad de NendoU de Nsp15 es responsable de la interferencia de proteínas con la respuesta inmune innata

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quisiera que trabajes en el desarrollo de inhibidores contra el proteasome (nanomaquina que degrada proteinas) de Tuberculosis.

le dire a gustavo que pueda enseñarte a hacer VS y redocking, la simulación ya la sabes hacer,

el codigo de la proteina y su inhibidor es &OCW

6OCW