# data[].attributes.headline
Immunochemical engineering of cell surfaces to generate virus resistance

# data[].attributes.subject
Immunochemical engineering of cell surfaces to generate virus resistance

# data[].attributes.attachment_link.provider_name
Proceedings of the National Academy of Sciences

# data[].attributes.attachment_link.readable_text
1. aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

2. bDepartment of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037;

3. cDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;

4. dShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China

Contributed by Richard A. Lerner, March 14, 2017 (sent for review February 17, 2017; reviewed by Raymond A. Dwek, Dario Neri, and David I. Watkins)

Significance

A method that renders cells resistant to virus infection is reported. The method is based on anchoring antibodies to cell surface virus receptors to the plasma membrane. Because of their effective molarity, the antibodies are much more effective than if they were free in the circulation. The antibodies interact with the receptors and block virus attachment. In infections with viruses such as HIV, cells rendered resistant to infection will be selected over infected cells, thereby opening the possibility of curing the infection.

Abstract

Modern immunochemical engineering allows the creation of cells that either secrete antibodies or incorporate them into various cellular compartments, including the plasma membrane. Because the receptors for most viruses are known, if one can achieve the proper stoichiometry and geometry, plasma membrane-associated antibodies to these receptors should block viral infection. In this report, we test this concept for two different viruses, human rhinovirus and HIV. Plasma membrane-tethered antibodies efficiently rendered cells permanently nonpermissive for infection by both these viruses. Membrane-bound antibodies were much more efficient than free antibody in preventing infection, likely because of the effective molarity of membrane bound antibodies. Such resistant cells may restore immune-competence to otherwise compromised HIV patients.

Results

Rhinovirus.

Ultimately, we wished to study the possibility that membrane-bound antibodies could completely protect the host cell from virus infection for both enveloped and nonenveloped viruses. We first studied rhinovirus as our model system because it is quite simple and robust and is easily handled without biosafety concerns. We targeted the ICAM protein because it is known to be the rhinovirus A and B receptor ( 4). Because the structure of the ICAM protein in complex with membrane-tethered antibodies is not known, we used a computer simulation to predict the optimum geometry for the interaction of the two proteins (). The total length of the antibody plus linker () is approximately 190 Å, which is similar to the simulated full length of the ICAM protein. According to their size, three possible interaction models were proposed. They could both flex and move toward each other, or if ICAM itself is too rigid to bend, the antibody could bend to achieve contact because the linker is flexible. Alternatively, they could be brought into proximity by membrane curvature in the microdomains or trafficking vesicles (). Because rhinovirus uses domain 1 located on the top of the receptor for docking, one should be able to select a functional anti-ICAM from combinatorial antibody libraries by using protection from virus induced cell death as the selectable phenotype ( 5).

Screening for ICAM-1 Binding Antibodies That Prevent Infection.

We initially selected antibodies that simply bind to the ICAM protein by using a combinatorial human antibody library in phage with a diversity of >10 ⁹ members. After two rounds of panning against the ICAM protein, the antibody encoding genes from the recovered phage particles were transferred to lentivirus vectors that were designed to deliver the gene to mammalian cells and express the antibody protein tethered to the plasma membrane (). The lentiviral vectors harboring the selected antibody encoding fragments were used to package a pool of lentivirus as the input library, and HeLa cells were infected at a MOI = 2 for the functional selection. The ability of lentivirus to express membrane-tethered proteins in this system was confirmed by using the florescence tomato protein in the place of the antibody fragment (). The key finding is that the membrane-tethered protein covers the entire cell surface, indicating that when the MTA is expressed no trajectory for successful viral infection remains. To give more quantitative information, we used Western blotting versus antibody standards to estimate the number of MTA molecules linked to the plasma membrane of HeLa cells. There are approximately 30,000 MTAs expressed on the surface of each cell.

Two days after infection with the pool of antibodies in lentivirus, rhinovirus was added to the HeLa cell culture at MOI = 1. The vast majority of cells died after 2 d as is normally seen. However, some virus-resistant colonies started to form and expand even in the presence of a high density of rhinovirus particles (). Importantly, these studies showed that a selection based on cell survival can yield cells that are completely protected from cell death, presumably because of the MTAs linked to their surface.

All of the resistant colonies were collected for preparation of genomic DNA. The antibody-encoding fragments were recovered by PCR and then cloned again in the lentiviral vector. Direct comparison between the antibodies selected because they protect from infection (output pool) and the antibodies that were only selected for binding (input pool) showed that the output pool was much more effective in preventing infection. As a control, it was shown that lentivirus that only expressed tomato did not protect. ().

To generate individual antibodies, 100 bacterial clones were picked from the output library and sequenced. They were individually packaged into lentivirus, delivered to HeLa cells, expressed on the surface, and tested for protection from infection. Five different antibodies were identified, Ab03, Ab28, Ab51, Ab83, and Ab100. When examined 2 and 3 d after rhinovirus infection, all of them showed persistent protection, although to slightly different extents (). Interestingly, when expressed in the soluble format, the five antibodies demonstrated different abilities to inhibit rhinovirus infection (), with Ab100 as the strongest and Ab51 the weakest. Among different antibodies, the relationship between the degrees of protection by each soluble antibody at the highest concentration tested compared with the same antibody in the MTA format follows an exponential function, indicating the important role of effective molarity for the MTAs (). Thus, the protective effect of a given antibody is vastly improved when it is used in the MTA format. It is important to note that the number of cells bearing MTA after an initial decrease in number actually increase over time, ultimately reaching the virtually 100% survival seen for uninfected cells (, red circle). Because this increase in cell number occurs in the presence of virus, the initial killing followed by emergence of resistance has all of the features of a process based on selection.

Selecting Antibodies That Preserve Normal Receptor Function by Paratope Fine-Tuning.

ICAM is known to mediate T-cell binding to endothelial cells by forming a complex with LFA. Because ICAM binding to LFA and rhinovirus use ICAM domain 1, there is a possibility that the rhinovirus blocking antibodies may interfere with the normal physiological functions of ICAM. This interference has been observed in other virus systems. For example, many of the CD4-binding antibodies block HIV entry efficiently but also comprise immune function that limits their clinical utility. Thus, we wished to test whether our antibodies inhibit normal ICAM1 function. A T-cell attachment assay was conducted as previously described except that we used high-content imaging to get a more accurate absolute count of the interacting T cells. At high concentration, the antibody that is most potent in protecting from virus infection (Ab100) is also the most efficient in blocking T-cell attachment, whereas other antibodies have more variable effects on T-cell binding ().

We thought that adjusting the paratope of the ICAM-1 binding antibodies might fine-tune the recognition mode, thus influencing retention of normal ICAM function ( Fig. S1). Hence, we selected germ-line hotspots in Ab100 for generating a library of mutant antibodies. Because CDRH3 generally contributes more to antigen binding than CDR1 or CDR2, only hotspot residues in CDR3 of both light and heavy chains were chosen for mutagenesis. The library was designed to encode 10%∼50% of the wild-type amino acids. The other 19 amino acids had as many mutations as possible at each targeted position by the use of degenerate codons. We generated a library with a diversity of 2 × 10 ⁸ in a yeast surface expression vector, pCTcon2. The screening procedure resembles standard yeast display, combining magnet-assisted cell sorting (MACS) and five rounds of flow cytometry-assisted cell sorting (FACS) to select for antibodies with the highest affinity (). After five rounds of selection, 20 clones were picked from the output yeast pool and 3 were selected for further study based on their unique sequences (). The K D for binding to ICAM by the newly selected antibodies was, 2.50 nM, 2.03 nM, and 3.67 nM, respectively for HA02, LA03, and LA06 compared with 6.36 nM for the original Ab100 (). The antibodies were subsequently assayed for their ability to protect in vitro against rhinovirus infection. 84H10, a control antibody that is known to block ICAM function was determined to have an IC ₅₀ of 9 nM, whereas Ab100 has an IC ₅₀ of 2.4 nM. The mutant antibodies' IC ₅₀ for blocking infection are 1.7 nM, 0.7 nM, and 0.5 nM for HA02, LA03, and LA06, respectively ().

The T cells' attachment assay of these mutant antibodies using analysis by high-content imaging showed that HA02 and LA03 at high concentration only blocked T-cell attachment by 30%, whereas blockage by LA06 was nearly complete (). Thus, it appears that one can select antibodies that retain their ability to neutralize virus infectivity, whereas preserving the normal function of the target receptor. For example, antibody from clone 83 has an IC ₅₀ for prevention of virus infection of approximately 10 nM, whereas it completely spares T-cell attachment at 100 nM.

Blocking HIV Infection by Membrane-Tethered Antibodies.

Encouraged by the results in the rhinovirus system, we turned to HIV. The idea was to determine whether MTAs against known HIV cellular receptors could render the cells resistant to infection. To test this concept, we studied anti-CD4 antibody, whichis known to be a receptor for HIV. We engineered TZM-bl cells to express anti-CD4 ScFv antibodies in a MTA format. These cells are widely used to evaluate antibody neutralization in standardized HIV-1 infectivity assays because the degree of virus neutralization in this assay correlates well with protection in nonhuman primates. The anti-CD4 antibody leu3a (converted to scFv) in a MTA format demonstrated almost complete inhibition of infectivity for four of six viruses (92RW020, 92BR020, JRFL, BG505) in a cross-clade virus panel and significant protection for the remaining two viruses (94IN108 and HXB2). Again, by comparison, the soluble version of the antibody was either nonneutralizing or poorly neutralizing even at a high concentration of 50 μg/mL ( Fig. 6). The results of these studies again show that expression of antibodies on the cell surface of target cells results in enhanced neutralization activity compared with soluble antibody.

Finally, in terms of stoichiometry, the number of expected MTA molecules per cell is in the range of the number of known HIV receptors on T cells ( 6). The absolute concentration of receptors appears to be critical for HIV infection. For example, for CCR5, when the receptor threshold falls below 10,000 molecules per cell, the ability to infect cells falls dramatically ( 7). Thus, MTAs can function by simply lowering the number of virus receptors below a critical density.

Discussion

In this study, we show that plasma membrane-tethered antibodies to virus receptors can inhibit entry of two prototype viruses, the nonenveloped rhinovirus and the larger enveloped HIV. Although antibody engineering of cell surfaces appears to be a promising approach to generating cells that have the potential to render the host resistant to virus infection, attention will inevitably turn to the most desirable properties of the antibody. This selection of antibodies happens in all cases where one ponders the use of antibodies to contain an infectious process whether it is by common vaccination or the special case of cell engineering as studied here. In our case, the most desirable antibody should have high affinity for the receptor and not interfere with the normal physiological function of the receptor. Toward this end, a major advantage of our approach is that it is not limited to receptors without critical functions because antibodies can be selected that block virus infection while sparing the native function of the receptor. Although such antibodies might be rare, nowadays we have technologies to select such rare antibodies from vast numbers of candidates in combinatorial libraries. Also, one might simultaneously target multiple receptors and coreceptors. The choice of the target receptor is, of course, important with CCR5, CD4, and CXCR4 being obvious choices that could be used either individually or in combination. Importantly, the efficiency of protection need not be absolute because one relies on the process of selection when the resistant cells are returned to the host. Thus, the T cells that are completely resistant to infection should expand because they have a growth advantage and could thereby lead to sustained resistance to HIV infection. Initially one expects the host to be a chimera of resistant and nonresistant cells, but over time the pool of resistant cells should become the majority.

Thus, while we have proof of concept, the exact antibodies studied here are unlikely to be the ones ultimately used in clinical trials. This selection of antibodies is similar to what has already happened in the field with anti-gp120 antibodies because the community continues to discover ever better antibodies both in terms of potency and range of neutralization ( 8). Finally, we may wish to make the antibody gene inducible so as to be able to generate resistant cells at will. Indeed, when the fluorescent protein tomato is integrated into the lentiviral MTA construct under the control of a tetracycline-inducible promoter, the fluorescent signal on the surface is absent but can be switched on by adding doxycycline to the culture media ( Fig. S3). Thus, the next steps toward a therapeutic regimen will involve selection of the best antibody in terms of specificity and potency followed by studies in primates.

There are, of course, other approaches to the generation of cells resistant to HIV infection. One widely discussed approach is the use of gene editing to eliminate integrated virus(s) or virus receptors. When it comes to integrated viral genomes, a major problem with gene editing is that the editing process accelerates the generation of replication-competent viral escape mutants that are now resistant to Cas9/sgRNA, thereby greatly limiting its potential for HIV therapy ( 9). Because our approach is not genetic, viral escape would have to involve development of an alternative route for virus entry into cells that is unlikely and, if it happened, would be expected to be highly inefficient and selected against at the population level.

Because patients with null mutations in CCR5 (CCR5 Δ32) may be resistant to HIV infection, another approach is to use gene editing to disable the CCR5 receptor ( 10, 11). However, the long-term consequences of having CCR5 defective alleles is not known, particularly when it comes to the importance of this chemokine receptor in combating other virus infections ( 12). This potential need to maintain the natural function of the receptor is one reason that we may wish to have inducible gene expression of the MTA so as to allow restoration of the receptor in certain clinical situations. Also, in some cases, chronically infected patients who received CCR5 Δ32 stem cell transplants suffered virus outbreak because of selection in vivo of CXCR4-tropic viruses ( 13). Again, this need for the receptor is why we may wish to use more than one MTA, particularly inclusive of an antibody against CXCR4 that allows retention of its critical physiological function.

Finally, it is of interest to place our work in perspective relative to approaches using vaccination, passive antibodies, or small molecule drug therapy. All these approaches are aimed at HIV itself, whereas MTAs target the host and work in conjunction with the process of selection. Basically, the goal comes down to the initiation of a process of real-time Darwinian selection in the host. Paradoxically, the virus becomes the driving force for selection of a resistant host who no longer harbors virus. For this selection to happen, one may need intervals such as "drug holidays" where viral replication is allowed, thereby using the power of selection to help eliminate reservoirs of infected cells.

SI Materials and Methods

Production of Lentivirus in HEK 293T.

Lentivirus was produced by reverse transfection of 3.6 × 10 ⁶ HEK 293T cells in a six-well plate, using 1.5 μg of lentiviral expression vector from the pooled anti-ICAM1 antibodies, 0.6 μg of pVSV-G and 0.8 μg of pCMV-dR8.9, and 7.5 μL of Lipofectamine 3000 (Thermo Fisher). After incubation at 37 °C for 6 h, medium was replaced with fresh DMEM with 10% FBS (D10F). Forty-eight hours after medium exchange, cell supernatant was centrifuged at 931 × g for 10 min at room temperature and collected. Cellular debris was further removed by a 45-μm pore filter. Lenti-X p24 rapid titer kit (Clontech) was used to determine the p24 level of lentivirus so that host cells can be transduced at a specific MOI. The virus preparations were aliquoted and frozen at −80 °C.

Mutant Library Construction for Affinity Maturation.

Germ-line hotpots were targeted for mutagenesis, as described ( 14). In short, RGYW/AGY mutational hotspots in CDR3 of clone IV100 were randomized by degenerate PCR mutagenesis. A mutant library was constructed by in vivo homologous recombination between the PCR products and linearized yeast display vector pCTCon2 ( 15). The library was transformed into the yeast strain EBY100 and grown in SD-CAA overnight at 30 °C. Following induction in SGR-CAA at 20 °C for 20 h with a starting OD ₆₀₀ ∼ 1, the yeast library was subjected to one round of MACS and five rounds of FACS.

MACS and FACS.

MACS was used for the primary library to reduce library diversity. The galactose-induced yeast cells were harvested and incubated with 50 nM of biotinylated human ICAM-1 at room temperature (RT) for 1 h. After washing and incubation with Streptavidin MicroBeads (Miltenyi Biotec) at RT for 30 min, ICAM-1 binding yeast-display scFv were then enriched by magnetic sorting over an LS column (Miltenyi Biotec). Recovered yeast were expanded in SD-CAA overnight at 30 °C.

Round 1-2 of FACS screening were performed by incubation of 2.5 nM biotinylated ICAM-1 with scFv-displaying yeast for 1 h until equilibrium binding was reached. Chicken anti-c-Myc IgY (Thermo Fisher) and Alexa Fluor 488 Goat Anti-Chicken IgG (H+L) (Thermo Fisher) were used to determine scFv expression on the yeast cell surface. Anti-Biotin-APC (Miltenyi Biotec), DyLight 633 NeutrAvidin (Thermo Fisher) were applied respectively in each round for the assessment of antigen binding.

Biotinylated ICAM-1 (0.5 nM) was used for incubation for round 3-5 of FACS screening, with the use of R-Phycoerythrin Streptavidin (Thermo Fisher), DyLight 633 NeutrAvidin, and R-Phycoerythrin Streptavidin in each round. In round 4 and round 5, selection pressure was increased by off-rate selection. In short, after incubation with biotinylated ICAM-1 and buffer wash, nonbiotinylated ICAM-1 was added at a final concentration of 100 nM into the scFv-displaying yeast and incubated for 2 h to outcompete biotinylated ICAM-1 from weak binders. Cells were acquired on a BD FACSAria III (BD Biosciences), and data were analyzed by using FlowJo (Treestar).

PMA Jurkat Cell Adhesion Assay.

Human ICAM-1-Fc was coated onto a 96-well half-area high binding plate at 10 μg/mL PBS overnight at 4 °C. Plates were washed and blocked with 1% BSA in HBSS buffer for 1 h at RT. Antibodies were serially diluted with HBSS buffer containing 1% BSA and 2 mM MgCl ₂. The antibody solution was added to the plate (25 μL per well) and incubated for 15 min at RT. To each well was then added 25 μL of HBSS buffer containing 50 ng/mL PMA, 1% BSA, and 2 mM MgCl ₂. After 30 min, Jurkat cells were added to the plate (105 cells per well) in 100 μL of HBSS, 1% BSA and 2 mM MgCl ₂, and incubated at 37 °C/5% CO ₂ for 3 h. Cells attached on each well of the plate were imaged and quantified by using in Cell Analyzer 6000 following aspiration of the plate and washing with 200 μL of HBSS/2 mM MgCl ₂.

Pseudovirus Production and Neutralization Assays.

To produce pseudoviruses, plasmids encoding Env were cotransfected with an Env-deficient genomic backbone plasmid (pSG3∆Env) in a 1:2 ratio with the transfection reagent FuGENE 6 (Promega). Pseudoviruses were harvested 72 h after transfection for use in neutralization assays. Neutralizing activity was assessed by using a modified single round of replication pseudovirus assay and TZM-bl target cells, as described ( 16). Briefly, TZM-bl cells were seeded in a 96-well flat bottom plate at a concentration of 10,000 cells per well. For cell surface antibody display, the TZM-bl cells were infected with lentivirus for 48 h at 37 °C. For soluble antibody experiments, antibody was added to TZM-bl cells at the indicated concentrations and also incubated for 48 h. Pseudovirus was then added to these cells and incubated for an additional 48 h. Luminescence was then quantified 48 h following HIV pseudovirus infection via lysis and addition of Bright-GloTM Luciferase substrate (Promega).

Acknowledgments

We thank Dr. Rajesh Grover for helpful discussions.

Footnotes

Author contributions: J.X. and R.A.L. designed research; J.X., D.S., N.C.W., T.Z., and W.Z. performed research; J.X., D.S., D.R.B., and R.A.L. analyzed data; and R.A.L. wrote the paper.

Reviewers: R.A.D., University of Oxford; D.N., ETH Zurich; and D.I.W., University of Miami Miller School of Medicine.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1702764114/-/DCSupplemental.

# data[].attributes.attachment_link.title
Immunochemical engineering of cell surfaces to generate virus resistance

# data[].attributes.attachment_link.description
aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037; cDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; dShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China Contributed by Richard A.

# data[].attributes.headline
An electrochemical peptide-based biosensing platform for HIV detection- paper

# data[].attributes.subject
An electrochemical peptide-based biosensing platform for HIV detection- paper

# data[].attributes.attachment_link.readable_text
We have fabricated a potentially generalizable electrochemical peptide-based ( E-PB) sensor for the detection of HIV anti-p24 antibodies. The E-PB sensor is sensitive, specific and fares well even when challenged in a realistically complex medium such as human urine proxy.

# data[].attributes.attachment_link.title
An electrochemical peptide-based biosensing platform for HIV detection

# data[].attributes.attachment_link.description
We have fabricated a potentially generalizable electrochemical peptide-based ( E-PB) sensor for the detection of HIV anti-p24 antibodies. The E-PB sensor is sensitive, specific and fares well even when challenged in a realistically complex medium such as human urine proxy.

# data[].attributes.headline
News Article on tri-specific antibody treatment for HIV

# data[].attributes.subject
News Article on tri-specific antibody treatment for HIV

# data[].attributes.attachment_link.readable_text
An antibody that attacks 99 per cent of HIV strains has been developed by scientists for the first time.

The "exciting breakthrough" could eventually lead to treatment or even prevent transmission of the virus, with trials on humans due to get underway in 2018.

Experiments on 24 monkeys injected with HIV showed none developed the virus after first being given the new type of antibody, which attacks three critical parts of the disease.

The human immune system struggles to deal with HIV due to its ability to mutate and change appearance. Our bodies eventually become overwhelmed by the number of different strains of the virus.

But around one per cent of patients have the ability to develop "broadly neutralising antibodies". These bind to structures on the surface of the pathogens known as "spikes".

Spikes barely change and are identical among different strains, making it possible for these special antibodies to attack different mutations of the virus.

Now scientists have managed to combine three of these flexible antibodies into a powerful "tri-specific antibody".

Dr Gary Nabel, chief scientific officer at pharmaceutical company Sanofi and one of the report authors, told the BBC the results were "impressive".

"They are more potent and have greater breadth than any single naturally occurring antibody that's been discovered," he said.

"We're getting 99 per cent coverage, and getting coverage at very low concentrations of the antibody."

An estimated 36.7 million people worldwide were living with HIV or AIDS at the end of 2015, with the majority in sub-Saharan Africa. Of these, only 60 per cent of people are aware they have it.

In 2015 alone 1.1 million people died from the disease.

The study was published in the journal Science, and is a collaboration between the US National Institutes of Health and Sanofi.

The research included contributions from scientists at Harvard Medical School, The Scripps Research Institute and the Massachusetts Institute of Technology.

Reuse content

# data[].attributes.attachment_link.title
Scientists just created an antibody that attacks 99 per cent of HIV strains

# data[].attributes.attachment_link.description
An antibody that attacks 99 per cent of HIV strains has been developed by scientists for the first time. The "exciting breakthrough" could eventually lead to treatment or even prevent transmission of the virus, with trials on humans due to get underway in 2018.

# data[].attributes.headline
Research article on inhibition of T-cell proliferation to 'starve HIV'

# data[].attributes.subject
Research article on inhibition of T-cell proliferation to 'starve HIV'

# data[].attributes.attachment_link.readable_text
Harry E. Taylor,

Glenn E. Simmons Jr,

Thomas P. Mathews,

Atanu K. Khatua,

Waldemar Popik,

Craig W. Lindsley,

Richard T. D'Aquila,

H. Alex Brown

Subject Areas

For more information about PLOS Subject Areas, click here.

We want your feedback. Do these Subject Areas make sense for this article? Click the target next to the incorrect Subject Area and let us know. Thanks for your help!

Abstract

Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.

Author Summary

Replication of all human viruses depends on building blocks derived from the metabolic pathways of the infected host cell. The production of progeny virions requires synthesis of viral nucleic acids from deoxyribonucleotide triphosphates (dNTPs). HIV-1 infection in resting T cells is limited, at least in part, because the levels of critical nucleotides are low. However, stimulation of T cells turns on their metabolic machinery to increase c-Myc expression and subsequent synthesis of these key components of RNA and DNA, which augments HIV-1 replication. We have identified PLD1 as a key molecular switch that couples stimulatory T cell signals to c-Myc-dependent nucleotide biosynthesis. We also found that a small molecule that inhibits PLD1 suppresses HIV-1 replication by limiting c-Myc-dependent effects of T cell activation that support efficient HIV reverse transcription. Our study provides insight into a novel way of targeting T cell activation-induced processes such as nucleotide biosynthesis that has potential to augment current therapeutics for HIV-1.

Citation: Taylor HE, Simmons GE Jr, Mathews TP, Khatua AK, Popik W, Lindsley CW, et al. (2015) Phospholipase D1 Couples CD4⁺ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication. PLoS Pathog11(5): e1004864. https://doi.org/10.1371/journal.ppat.1004864

Editor: Susan R. Ross, University of Pennsylvania School of Medicine, UNITED STATES

Received: December 16, 2014; Accepted: April 7, 2015; Published: May 28, 2015

Copyright: © 2015 Taylor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research was supported in part by the National Institutes of Health National Institute of Mental Health (U54-MH084659) and the Vanderbilt Institute of Chemical Biology, Bixler-Johnson-Mayes Endowed Chair (HAB), and the William K. Warren Jr. Chair in Medicine (CWL). Vanderbilt University is a member of the National Institutes of Health Molecular Libraries Probe Production Centers Network and houses the Vanderbilt Specialized Chemistry Center for Accelerated Probe Development. WP acknowledges support from the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (5R21DK094735). HET and RTD gratefully acknowledge Northwestern Medicine and Northwestern University Feinberg School of Medicine institutional support for the HIV Translational Research Center, the Howard Taylor Ricketts Chair (RTD), and the Vanderbilt Institute for Clinical and Translational Research Scholar Award (5KL2RR024977) (HET) that enabled this work. This work was supported by the Northwestern University Flow Cytometry Facility and a Cancer Center Support Grant (NCI CA060553). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

HIV-1 replication in resting CD4+ T cells is restricted post-entry, but prior to integration [ 1]. Several groups have reported that suboptimal dNTP pools in these metabolically quiescent cells support only inefficient reverse transcription and subsequent integration [ 2, 3]. Cellular activation, or addition of exogenous deoxyribonucleosides, relieves the post-entry block to HIV-1 infection in resting CD4+ T cells [ 2, 3]. Decreasing dNTP pools in activated T cells with hydroxyurea (HU), a ribonucleotide reductase inhibitor, was also shown to suppress HIV-1 replication in vitro [ 4, 5], although clinical trials were limited by serious toxicities [ 6]. More recently, glucose metabolism has been identified to play a fundamental role in providing a carbon source for both T cell function and HIV-1 replication [ 7]. Notably, glucose uptake and its metabolism via the pentose phosphate pathway produces ribose intermediates that are critical for the synthesis of all nucleotides [ 8]. Expression of Glut1, a glucose transporter, is also essential for HIV-1 infection of activated CD4+ T cells [ 9]. Finally, catabolism of dNTPs is one of the mechanisms implicated in the anti-HIV activity of sterile alpha motif-histidine-aspartic domain-containing protein 1 (SAMHD1) in resting, but not activated, CD4+ T cells [ 1].

Recent reports have supported a prominent role of the c-Myc oncogene as a "master regulator" of transcriptional regulation of genes needed for nucleotide biosynthesis and glucose metabolism essential for both cellular and viral processes [ 10, 11]. In an elegant study utilizing acute conditional deletion of c-Myc in murine T cells, Wang and colleagues demonstrated that c-Myc is essential for metabolic reprogramming and nucleotide precursor accumulation in activated T cells [ 11]. Consistently, c-Myc was also found to be highly induced upon T cell activation and required for cell growth and proliferation [ 11]. Further, pharmacologic inhibition of the Ras/ERK pathway was found to abrogate expression of c-Myc after T cell activation [ 11]. Inhibition of either the Ras/ERK signaling module or c-Myc activity has been reported to suppress early steps of HIV-1 replication in activated T cells [ 12, 13, 14]. However, the mechanism by which T cell activation induces c-Myc expression to initiate this cascade remains undefined.

Interestingly, one pathway potentially involved in coupling T cell activation to c-Myc expression, phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine to choline and phosphatidic acid (PA) [ 15], is activated whether T cells are stimulated by the mitogenic lectin phytohaemagglutinin (PHA) or via antibody-mediated crosslinking of the T cell receptor (TCR). In humans, PLD exists as two isoforms derived from separate genes, PLD1 and PLD2 [ 16]. The two PLD isoforms have been implicated in a plethora of signaling pathways that influence numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis [ 16]. Furthermore, PA upregulates Ras/ERK [ 17], and increases expression of c-fos and c-Myc [ 18]. This occurs if PA is supplied either exogenously or endogenously through PLD1 or 2 activity [ 18]. Since PLD1 has been shown to mediate responses downstream of the T cell receptor [ 19], we tested the hypothesis that the PLD1 signaling pathway couples T cell activation to cellular processes essential for HIV-1 replication. Experiments undertaken here using pharmacologic and genetic inhibition of PLD1 provide evidence that PLD1 activity links T cell activation signals to the Ras/ERK/c-Myc signaling cascade required for metabolic reprogramming that expands dNTP pools. We also report that PLD1 inhibition blocks HIV-1 reverse transcription and replication.

Results

Phospholipase D1 inhibition blocks the c-Myc-dependent dNTP biosynthetic transcriptional program

Human resting CD4+ T cells were stimulated with PHA in the presence and absence of a PLD1-selective small molecule inhibitor, VU0359595 (PLD1i) [ 20]. Here, inhibition of PLD1 reduced ERK1/2 phosphorylation after T cell activation with PHA, in a similar manner as direct suppression of ERK activity with the selective MEK/ERK inhibitor U0126 (p-ERK in Fig 1A). Ras/ERK signaling also promotes site-specific phosphorylation of ribosomal protein S6 (S6) at Ser 235/236 (p-S6, in Fig 1A) [ 21]. Notably, resting CD4+ T cells activated with PHA in the presence of either PLD1i or U0126 had a marked reduction in S6 phosphorylation (p-S6, Fig 1). Since PLD1 has been shown to activate the parallel pathway of mechanistic target of rapamycin (mTOR) / S6 kinase 1 (S6K1), perturbation of mTOR by PLD1i to decrease its activity could also contribute to a diminution in p-S6. To test this possibility, the levels of phosphorylation of specific targets of the mTOR pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase (CAD) at Ser ¹⁸⁵⁹ (p-CAD) and translation repressor protein 4E-BP1at Ser ⁶⁵ (p-4E-BP1) were also determined. PHA stimulation increased the abundance of p-S6, p-CAD and p-4E-BP1, while rapamycin, an allosteric mTORC1 inhibitor known to suppress c-Myc expression, blocked these phosphorylation events ( Fig 1A) [ 11]. Inhibition of either ERK or PLD1 also reduced the levels of p-S6, p-CAD, and p-4E-BP1, suggesting that PLD1i does indeed suppress mTOR activity in stimulated CD4+ T cells. PLD1i also abrogated induction of the total level of these proteins following T cell activation (S6, CAD, 4E-BP1; Fig 1A). Since depletion of c-Myc decreased levels of total S6, CAD, and 4E-BP1 [ 11, 22], we hypothesized that PLD1 inhibition diminished the overall levels of these proteins by decreasing c-Myc expression. Therefore, we assessed expression levels of c-Myc in cells that were stimulated with PHA in the presence of PLD1 and ERK inhibitors. Levels of the c-Myc-dependent nucleotide biosynthetic enzymes thymidylate synthase (TS), large subunit of ribonucleotide reductase (RRM1), and small catalytic subunit of ribonucleotide reductase (RRM2) were also studied. Blocking PLD1, ERK, or mTOR impaired activation-dependent induction of c-Myc, TS and RRM2 ( Fig 1A), as well as RRM1, proteins ( S1 Fig). These results phenocopied the effects of a specific c-Myc inhibitor (10058-F4) (Myci in Fig 1A) [ 23]. PLD1i and Myci also inhibited activation-induced expression of RRM2 ( Fig 1B) and RRM1 ( S1 Fig) in primary CD4+ T cells. To confirm genetically that PLD1 is required for optimal c-Myc expression, ERK and mTOR activity in activated CD4+ T cells, we depleted PLD1 by siRNA-mediated silencing. We observed reduced expression of c-Myc, as well as reduced phosphorylation of ERK and mTOR target 4E-BP1 with two independent PLD1 siRNAs ( Fig 1C). c-Myc depletion by siRNA also reduced expression of PLD1, as well as known c-Myc dependent targets ( Fig 1C). These results suggest that PLD1 catalytic activity couples T cell activation signals to de novo nucleotide biosynthesis by augmenting ERK and mTOR-dependent c-Myc expression. These results are consistent with the effects of PLD1i here and those previously reported [ 24]. Taken together, these results suggest a positive correlation between the level of c-Myc and PLD1expression in activated CD4+ T cells consistent with a positive feedback loop ( Fig 1D). We hypothesize that PLD1 activity increases expression of both c-Myc and previously described c-Myc-dependent genes, and that increases in PLD1 and c-Myc amplify each other's expression ( Fig 1D).

Stimulation of CD4+ T cells with PHA/IL2 for only 30 min was sufficient to induce a 2-fold increase in PLD activity, relative to the unstimulated control (2.07 ± 0.33 vs. 1 ± 0.49 relative fluorescence, P = 0.023). Importantly, this rapid burst of activity after T cell activation was reduced by PLD1i pretreatment, when compared to PHA/IL-2 stimulated cells (1.27 ± 0.35, P = 0.044), but was not significantly different when compared to unstimulated cells ( P = 0.51). Others have documented that c-Myc expression is also rapidly induced, within 2 hours of T cell activation [ 11]. This may help explain why effects of PLD1i on c-Myc ( Fig 1C) appeared more robust than those of siRNAs against PLD1 ( Fig 1A). Maximal effects of siRNA on protein expression are not observed before 24 hours after transfection. Therefore, the greater decrease of c-Myc observed here with PLD1i than siRNA against PLD1 is consistent with more acute inhibition of PLD1 by the inhibitor than genetic silencing [ 11, 25].

PLD1 activity is required for normal expression of Glut1 and nutrient transporters in activated T cells

c-Myc also drives the expression of key nutrient transporters needed for cell growth and proliferation after T cell activation: Glut1 (for glucose); SNAT1 and SNAT2 (for glutamine); Slc7a5/LAT1 (for large neutral amino acids) [ 11]. Since CD28 stimulation is essential for optimal surface expression of Glut1 [ 26], resting CD4+ T cells were activated with anti-CD3/anti-CD28. Cells were then surface-stained for Glut1 and the activation marker CD25. Consistent with previous reports, inhibition of PLD1 suppressed expression of CD25 [ 27] ( Fig 2A); however, PLD1i had little observable effect on expression of activation markers CD71 or CD98, suggesting that PLD1i does not lead to global inhibition of T cell activation ( S2 Fig). PLD1i treatment prevented the upregulation of Glut1 surface expression on a sub-population of activated T cells, similarly to prior observations ( Fig 2A) [ 28]. Additionally, we found that inhibition of PLD1 or c-Myc in activated CD4+ T cells reduced total cellular expression of nutrient transporters Glut1, SNAT1, and SNAT2. Importantly, inhibition of upstream mediators of c-Myc expression (ERK and mTORC1) also impaired expression of these nutrient transporters ( Fig 2B). Quantitative PCR (qPCR) on RNA from CD4+ T cells transfected with siRNAs targeting c-Myc or PLD1 confirmed reduced mRNA expression of RRM2, SNAT1, SNAT2, and Slc7a5/LAT1 ( Fig 2C). Slc7a5/LAT1 is required for both mTORC1 activity and c-Myc expression in activated T cells ( Fig 2C) [ 29]. Knockdown of c-Myc and PLD1 again resulted in reduced expression of the alternate RNA ( Fig 2C), consistent with data in Fig 1C that suggested a positive feedback loop as illustrated in Fig 1D. The observations of inhibition of PLD1 activity resulting in the impairment of coordinated expression of c-Myc, nucleotide biosynthetic genes, and nutrient transporters are consistent with PLD1 signaling being upstream of induction of c-Myc in activated T cells.

Inhibition of PLD1 activity limits activation-induced T cell proliferation

Activation of T cells leads to increased synthesis of biosynthetic precursors that enable cell proliferation [ 11]. To this end, c-Myc coordinates increased uptake of glucose and glutamine with nucleotide biosynthesis to facilitate metabolic reprogramming of activated CD4+ T cells. Furthermore, like genetic ablation of c-Myc activity, glucose or glutamine starvation severely compromises activation-induced proliferation of T cells [ 11]. Since inhibition of PLD1 activity also reduces both c-Myc ( Fig 1) and c-Myc-dependent nutrient transporter expression ( Fig 2), we investigated the effects of PLD1i on cell cycle distribution and proliferation of activated CD4+ T cells. First, CD4+ T cells were pretreated with indicated inhibitor and then stimulated for 72 h in the continued presence of inhibitor. Cell-cycle progression was determined by simultaneously staining for RNA (Pyronin Y) and DNA (7-AAD) followed by flow cytometry. Fig 3A shows the distribution of cell-cycle phases identified by this technique. Resting CD4+ T cells remain in G ₀, but increase their RNA content after stimulation and progress into G 1a then G 1b. Activated CD4+ T cells then initiate DNA synthesis and enter S phase followed by G2/M phase completion. We found that PLD1i-treated cells progressed to all stages of the cell-cycle; however, when compared to control cells, more PLD1i-treated cells were in G 1b (24.5% versus 16.8%) ( Fig 3B). This suggested that inhibition of PLD1 activity delayed the initiation of DNA synthesis at the G 1b/S boundary. Consistent with this hypothesis, genetic ablation of RRM2, a c-Myc target gene suppressed by PLD1i ( Fig 1A), was previously found to induce G1/S phase cell-cycle arrest [ 30]. We also directly assessed proliferation of PLD1i-treated CD4+ T cells by determining the dilution of CellTRACE Violet stain by flow cytometry 72 h after stimulation ( Fig 3C). PLD1i suppressed activation-induced T cell proliferation in a concentration-dependent manner, albeit less so than did the c-Myc inhibitor (10058-F4). We also observed a delay of activation-induced proliferation by both U0126 and rapamycin ( Fig 3C), as previously reported [ 31, 32]. Cytotoxicity was not detected with PLD1i ( S2 Fig, bottom panel).

PLD1 activity is essential for dNTP pool expansion in activated CD4+ T cells

To directly assess dNTP pool expansion, resting CD4+ T cells were stimulated with PHA/IL2 in the presence and absence of PLD1i, and dNTP levels were quantified by mass spectrometry. PHA stimulation of CD4+ T cells resulted in 3.66-, 1.6-, and 9-fold increase in dATP, dCTP, and dTTP, respectively. Inhibition of PLD1 activity potently restricted the expansion of the dNTP pools. Increases in both dATP and dCTP were nearly completely inhibited and dTTP levels only increased 2-fold in the presence of PLD1 inhibitor (Fig 4A- 4C). Hydroxyurea (HU) treatment decreased only dATP levels ( Fig 4A), as previously reported [ 33].

Restriction of dNTP pool expansion by PLD1 inhibition contributes to suppression of HIV-1 replication in activated T cells

Since inhibition of PLD1 activity in activated CD4+ T cells limits dNTP pool expansion, we hypothesized that HIV-1 replication would be impaired. To test this hypothesis, resting primary CD4+ T cells were pretreated with PLD1i or vehicle and then stimulated with PHA/IL2. Cells were then infected with a single-round CXCR4-tropic envelope-pseudotyped GFP-expressing HIV-1. CXCR4-tropic virus, rather than CCR5-tropic virus, was used to limit assessment to effects of PLD1i on post-entry steps of HIV-1 replication. This is because PLD1i-mediated decreases in mTOR activity that diminish CCR5 surface expression and HIV-1 entry could confound analyses of CCR5-tropic virus [ 34]. PLD1i inhibited CXCR4-tropic HIV-1 infection by nearly 75% in CD4+ T cells from four independent donors ( Fig 5A). The effects of PLD1i on HIV infection were rescued by adding exogenous deoxyribonucleosides (dN), which bypassed the need for ribonucleotide synthesis and reduction; degree of rescue varied in cells from different donors ( Fig 5A). Exogenous dN had little effect on HIV-1 infection in control cells ( Fig 5A). Quantitative PCR was used to measure viral early reverse transcripts (ERT), late reverse transcripts (LRT), and 2-LTR circles at 24 hours after infection ( Fig 5B); the latter is an indicator of nuclear import of full-length viral cDNA. PLD1i had little effect on the level of ERT cDNA, consistent with normal levels of HIV-1 cell entry and initiation of reverse transcription ( Fig 5B). Assessment of CD4 and CXCR4 surface expression on PLD1i-treated cells also confirmed lack of receptor or co-receptor down-regulation by PLD1i that could affect entry (S4 Fig). PLD1i suppressed the accumulation of LRT cDNA after HIV-1 infection ( Fig 5B), consistent with a previous study of ERK inhibitors [ 13]. PLD1i also reduced the levels of 2-LTR circles more markedly than LRT cDNA. Treatment of cells with HU, known to limit HIV-1 reverse transcription and dNTP pools by inhibiting ribonucleotide reductase RRM2-dependent activity, also reduced the levels of LRT and 2-LTR circles in HIV-1 infected cells, with a similarly greater effect on 2-LTR circles ( Fig 5B)[ 5].

To confirm and further define the requirement of PLD1-dependent processes for HIV-1 replication, in a separate experiment we determined the effects of PLD1i on the accumulation of viral cDNA products at 8, 16, and 24 h after infection ( Fig 5C). Consistent with PLD1i-dependent effects on dNTP pools, the kinetics of reverse transcription was markedly delayed when compared to DMSO vehicle-treated cells. Reduced levels of LRT, and 2-LTR cDNA were again detected in PLD1i-treated cells at each time point. ERT cDNA levels were decreased at 8 and 16 hours, but only minimally decreased at 24 hours. Furthermore, inhibition of the PLD1 target c-Myc recapitulated these effects at the 24 h time point (other time points not studied with Myci) ( Fig 5C). This effect on the kinetics of reverse transcription can cause a "bottle-neck" upstream of HIV-1 nuclear import and can explain, at least in part, the reduction in 2-LTR circle levels we observed in PLD1i-treated cells based on a delay in availability of completed reverse transcripts in the cytoplasm.

Since PLD1i-treated cells have reduced dNTP pools and exogenous dN have been shown to increase the kinetics of reverse transcription in resting CD4+ T cells [ 3], we determined the effects of dN addition on HIV-1 cDNA products in PLD1i-treated cells ( Fig 5D). Exogenous dN increased the levels of LRT cDNA in PLD1i-treated cells, consistent with PLD1i's mechanism of RT inhibition being due to its effects on dNTP pool expansion. Interestingly, dN addition did not reverse PLD1i-decreased 2-LTR formation ( Fig 5D). This observation suggests that inhibition of PLD1 activity has an additional effect, not reversed by exogenous dN that diminishes HIV-1 cDNA nuclear import and/or 2-LTR formation in the nucleus.

Discussion

This study demonstrates that PLD1 is required to couple activation of primary CD4+ T cells to the c-Myc-dependent coordinated upregulation of nutrient transporters, dNTP biosynthesis, and other biosynthetic pathways that we and others have previously reported to support HIV-1 replication [ 35]. Our results now also suggest a positive feedback loop between c-Myc and PLD1 not previously appreciated ( Fig 1D). Loss of PLD1-mediated metabolic reprogramming caused dNTP-dependent delay in the accumulation of HIV-1 late reverse transcripts and other anti-HIV effects. PLD1-dependent downstream effects are also likely to be critical for replication of cells and other viruses, since c-Myc overexpression increases accumulation of nucleotides critical for DNA replication and cell division of cancer cells and adenovirus-infected cells [ 36, 37].

Addition of exogenous dN rescues the PLD1i-mediated decreases in HIV-1 replication and accumulation of late reverse transcripts ( Fig 5). This is evidence that PLD1i acts against HIV-1 through a specific effect that limits dNTP pool expansion following T cell activation, rather than via an off-target effect. It is of note that siRNA against PLD1 did not block HIV-1 reverse transcription in our hands. However, the demonstration here, and elsewhere, of rapid onset of PLD activity following T cell activation (in 30 minutes) indicates a technical limitation in using genetic silencing to confirm the specificity of the potent and very rapid effects of PLD1i, given that siRNAs do not decrease protein expression until 24 hours after T cell activation [ 25]. Results also show that limited dNTP pool expansion is not the only mechanism by which PLD1i decreases HIV-1 replication. The greater decrease in 2-LTR circles than LRT (Fig 5B- 5D) and lack of reversal of the reduction in 2-LTR circles by added dN suggest that PLD1i has additional effects on nuclear import and/or 2-LTR circle formation that are independent of its effect on dNTP pools. In line with this postulate, it has been hypothesized that slowing reverse transcription may enhance the action of host cell restriction factors [ 38].

Toxicity profiles have been characterized and previously reported for the PLD2 preferring inhibitors [ 38], but to date detailed toxicological characterization has not been performed on the compound series showing preference for the PLD1 isoenzymes. Importantly though, compounds using the same chemical scaffold that were shown to be dual isoenzyme inhibitors, and similar to those used in this report, have been tested in human clinical trials and no overt toxicity was observed [ 39]. Given serious adverse events seen in clinical trials of HU-based regimens, it is important to further exclude potential toxicity of PLD1 inhibitors.

Glut1 expression in CD4+ cells is essential for HIV-1 replication in target CD4+ T cells, since knockdown of Glut1 inhibited early HIV-1 replication [ 9]. However, the mechanism through which Glut1 knockdown inhibited HIV-1 replication has not yet been delineated. The current results, and those previously reported, suggest the hypothesis that limiting both Glut1 and glutamine transporter expression may also indirectly decrease HIV-1 replication via host cell dNTP depletion. Moreover, increased Glut1 expression is observed in CD4+ T cells in HIV-infected patients and the magnitude of increase is directly associated with the pace of T cell depletion and disease progression, thus suggesting an additional rationale for targeting this pathway for therapeutic intervention [ 28].

The importance of expanded dNTP pools for HIV-1 replication is well established, and recent studies of SAMHD1 have added the suggestion that enhanced catabolism of dNTPs may also contribute to anti-HIV effects [ 39, 40]. Earlier reports have clearly shown that inhibiting ribonucleotide reductase activity with HU following PHA activation suppresses early steps of HIV-1 replication, established limiting host CD4+ T cell dNTP synthesis as an antiretroviral strategy [ 4, 5]. However, data shown in Fig 4 demonstrates that inhibition of PLD1-dependent biosynthetic pathways has a more robust effect on dNTP biosynthesis than RRM2 inhibition, the mechanistic target of HU. Taken together with results depicted in Fig 5, the data strongly support a mechanism where PLD-regulated nucleotide biosynthesis and other processes play a major role in supporting HIV-1 replication. We found that inhibition of PLD1 also limits CD4+ T cell activation-induced proliferation ( Fig 3C). Limiting proliferation of T cells may also benefit anti-HIV strategies in ways that medications targeting viral processes cannot. Abnormal T cell activation/proliferation is hypothesized to contribute to "non-AIDS" adverse outcomes, as it persists even among patients with prolonged suppression of HIV replication by current medications. Indeed, this excessive activation/proliferation may not be ablated even when antiretrovirals are started in the earliest stages of acute infection (Utay NS, et al. Abstract 47, CROI 2015, presented February 24, 2015). In addition, recent reports indicate that latently infected resting memory CD4+ T cells may persist during antiretroviral therapy at least in part, because of HIV integrant-driven cellular proliferation [ 41, 42]. If PLD1 inhibition is found to be safe in the future, it could be used to test if these pathogenic processes can be ameliorated. Importantly, certain PLD inhibitors also have demonstrated ability to traverse the blood brain barrier to target HIV-1 replication in myeloid-derived cells in the CNS, unlike some current anti-HIV drugs [ 43, 44]. Furthermore, perturbation of nucleotide pools may be an additional factor beyond recently described effects on innate and adaptive immune responses contributing to PLD inhibitor-mediated blockade of influenza virus replication [ 45]. It is also provocative to speculate that short-term blockade of host cell synthesis of ribonucleotides and deoxyribonucleotides may provide a strategy for broad-spectrum activity against diverse RNA and DNA viruses. When compared to the anti-HIV activity of FDA-approved antiretrovirals, the effects of PLD1i are modest; however, this inhibitor constitutes only an early candidate in a search for more effective compounds for advancing to clinical development. Further development of PLD inhibitors holds promise as a potential therapeutic for viral infections that require host nucleotide pools for replication as well as cancers, although the roles of PA production, whether biophysical, transcriptional, or as a signaling molecule, in these therapeutic interventions have yet to be fully elucidated.

Materials and Methods

Isolation and transfection of siRNA in primary human resting CD4+ T cells

Peripheral blood mononuclear cells (PBMCs) were purified from healthy blood donor specimens obtained from Lifesource (Rosemont, IL) by Ficoll-Hypaque PREMIUM (GE Healthcare) gradient centrifugation. Resting CD4⁺ T cells were isolated from negatively-selected total CD4⁺ T cells (CD4⁺ T Cell Isolation Kit, Miltenyi Biotec) using CD25+ and HLA-DR+ microbeads (Miltenyi Biotec) and cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), glutamine (2 mM) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin). Cells were activated with PHA-L (5 μg/ml) (Roche) and IL-2 (20U/ml) (Roche) or anti-CD3/anti-CD28 beads (Invitrogen) (1 bead/5 cells).

For transfections, nontargeting or siRNAs targeting c-Myc (Santa Cruz Biotech) or PLD1 (Santa Cruz Biotech (PLD1 siRNA#1) and Dharmacon/ThermoFisher (PLD1 siRNA#2)) were nucleofected into resting CD4+ T cells using an AMAXA nucleofector apparatus. Transfection was performed with human T-cell Nucleofector kit (LONZA), following the manufacturer's instructions. Briefly, 240 pmol (~3μg) of siRNA was added to 1 x 10 ⁷ cells resuspended in 100 μl of Nucleofector solution for each. Nucleofector program U-14 was used. Nucleofected cells were transferred into 2 ml of medium and incubated at 37°C for 24 h before medium was changed and cells resuspended in 1 ml of medium. Cells were then stimulated by adding anti-CD3/anti-CD28 beads (1:5; bead:cell ratio), cells and beads were pelleted for 5 minutes at 1200 x g in 96-U-well plates, and incubated at 37°C for 48 h before cells were harvested for analysis.

Flow cytometry

Cell cycle subcompartment determination by staining with 7-aminoactinomycin D (Invitrogen) and pyronin Y (Sigma) was performed as previously described [ 46]. For analysis of surface markers, cells were stained at 4°C for 30 min with antibodies against Glut1 (R&D Systems), CD25 (BD Bioscience), CD71 (BD Bioscience), and CD98 (BD Bioscience) in PBS containing 1% BSA. Flow cytometric data was obtained on a LSRFortessa (Becton Dickinson) and analyzed with FlowJo software (TreeStar).

Cell proliferation

To follow cell division, cells (10 ⁷/ml) were pulsed with CellTRACE Violet (5μM) in PBS for 30 min at 37°C. Cells were then washed with PBS, resuspended in growth medium, and treated as indicated before stimulation by adding anti-CD3/anti-CD28 beads (1:5; bead:cell ratio). Cells and beads were pelleted for 5 minutes at 1200 x g in 96-U-well plates and incubated at 37°C for 72 h before cells were harvested for analysis by flow cytometry.

Virus preparation and viral infection

HIV-1 stocks were prepared by transfecting 293T cells as previously described [ 35]. All virus stocks were treated with TURBO DNase (Lifetechnologies) (100 U/ml) for 30 min at 37°C followed by 30 min at RT. CD4+ T cells were infected with virus (50 ng of p24 per 2 x 10 ⁵ cells) by spinoculation (1,200 x g, 2 h), followed by incubation at 37°C. Where indicated, cells were pretreated with PLD1i (VU0359595), 1mM HU, or 50 μM deoxyribonucleosides before infection.

Western blot analysis

Cells were washed with ice-cold phosphate-buffered saline, harvested and whole lysates were prepared using RIPA buffer [50mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1mM EDTA] with Protease Inhibitor Cocktail (Roche). Whole cell lysates were clarified (10,000 x g for 20 min at 4°C) and resolved by SDS-PAGE on 4-12% gradient Bis-Tris or 3-8% Tris-acetate polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with SuperBlock Blocking Buffer (Thermo Scientific) and incubated with indicated antibodies overnight at 4°C in SuperBlock. Blots were then incubated with anti-mouse or anti-rabbit antibody conjugated with horseradish peroxidase (Thermo Scientific) before detection (SuperSignal West Dura Chemiluminescent Substrate, Thermo Scientific).

PLD activity assay

Cells were treated as indicated and whole lysates were prepared using NP-40 lysis buffer [50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Nonidet P-40, and 1mM EDTA] with Protease Inhibitor Cocktail (Roche). Whole cell lysates were clarified (10,000 x g for 20 min at 4°C) and equal cell equivalents of whole cell lysates were used to determine total PLD activity with the Amplex Red PLD Assay kit (Lifetechnologies), according to the manufacturer's protocol.

Quantitative RT-PCR of c-Myc-dependent genes

Total RNA was isolated using RNeasy Plus Mini Kit (QIAGEN). Briefly, cDNA for qPCR was generated from total RNA using oligo dT primers (Promega) and M-MLV Reverse Transcriptase (Promega). Quantitative real-time PCR was performed on an iCycler (Bio-Rad) using iQSYBR Green (Bio-Rad) detection. Samples were analyzed in triplicate and normalized to actin RNA (ΔΔCt method). Primer pairs were: Actin (GGACTTCGAGCAAGAGATGG, GGACTTCGAGCAAGAGATGG), RRM2 (CAAGCGATGGCATAGTAA, TGTAAGTGTCAATAAGAAGACT), SNAT2 (AAGACCGCAGCCGTAGAAG, CAGCCATTAACACAGCCAGAC), LAT1 (GTGCCGTCCCTCGTGTTC, GCAGAGCCAGTTGAAGAAGC), PLD1 (TGTCGTGATACCACTTCTGCCA, AGCATTTCGAGCTGCTGTTGAA), c-Myc (TCCAGCTTGTACCTGCAGGATCTGA, CCTCCAGCAGAAGGTGATCCAGACT), ASCT2 (ATCGTGGAGATGGAGGA, AAGAGGTCCCAAAGGCAG), SNAT1 (GGCAGTGGGATTTTGGGACT, TGACCAAGGAGAACAACACCC).

Quantitative PCR for viral cDNA

Total cellular DNA was isolated from HIV-1 infected cells (DNeasy DNA isolation kit, Qiagen). Real time PCR was performed using iQSYBR Green (Bio-Rad) detection (Bio Rad CFX96). Reaction mixtures contained 250 nM of each primer and 100 to 300 ng template DNA in a final volume of 25 μl. The sequence of the primers used for real time PCR for early reverse transcription (ERT), late reverse transcription (LRT), two LTR circle DNA (2LTR) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were: ERT (TTA GAC CAG ATC TGC GCC TGG GAG, GGG TCT GAG GGA TCT CTA GTT ACC), LRT (TGT GTG CCC GTC TGT TGT GTG A, GAG TCC TGC GTC GAG AGA TCT), 2LTR (AAC TAG GGA ACC CAC TGC TTA AG, TCC ACA GAT CAA GGA TCT CTT GTC), GAPDH (GAA GGT GAA GGT CGG AGT, GAA GAT GGT GAT GGG ATT TC).

Samples were analyzed in triplicate and normalized to GAPDH (ΔΔCt method).

dNTP extraction and quantification by UPLC/MS/MS analysis

Cellular analysis of dNTPs was performed as previously reported [ 47, 48]. Briefly, after indicated treatments, cells were pelleted at 1000 × g, the supernatant aspirated and pellet resuspended in 500 μl of 70% methanol/water mixture at -20°C. Suspensions agitated for 4 minutes at 4°C, and then incubated at -20°C for one hour. Internal standards were then added; for dNTPs, 4 nmols of aminoallyl-UTP; for carbamoyl aspartate, 5 nmols of citrate- d ₄. Suspensions were agitated again at 4°C for one minute and centrifuged (18,000 × g, 10 minutes, 4°C). The supernatant was collected, transferred to a bullet tube and solvent evaporated under vacuum. Immediately prior to analysis, extracts were reconstituted in 100 μl of a 2 mM ammonium acetate, 3 mM hexylamine solution in water (pH 9.2).

dNTPs were quantified (adapted from [ 49]) by chromatography on Acquity I-class UPLC (Waters, Milford, MA) with detection by MDS SCIEX 4000QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems). Acquity BEH C18 column (2.1 x 50 mm, 1.7 μ) with a 10 μl sample injection was used for metabolite delivery and chromatographic resolution. Solvent A consisted of 2 mM ammonium acetate and 3 mM hexylamine in water (pH 9.2); solvent B was 100% acetonitrile. Flow rate of 0.6 ml/min was maintained with a linear gradient as follows: 0 minutes, 9% B; 2 minutes 16% B; 5 minutes, 16% B; 5.5 minutes, 100% B; 6.5 minutes, 100% B; 7 minutes, 9% B; 8 minutes, 9% B. For dNTP analysis, the mass spectrometer was operated in negative MRM mode; the following mass transitions were monitored: dATP, 490/159; dCTP, 466/159; TTP, 481/159. dGTP could not be reliably quantified with this method since its molecular fragmentation pattern and retention time were identical to ATP.

Statistical analysis

P values were calculated with Student's t test. P values<0.05 were considered significant.

Supporting Information

S1 Fig. PLD1 inhibition decreases the c-Myc-dependent dNTP biosynthetic gene expression.

(A) Western blot analysis of protein expression in whole cell lysates prepared from resting primary human CD4+ T cells that were treated as in Fig 1. (B) RRM1 mRNA levels in resting primary CD4+ T cells pretreated for 24h with DMSO vehicle, 100 μM c-Myci, or 10 μM PLD1i, then left resting or stimulated as in Fig 1 as determined by quantitative real-time PCR.
https://doi.org/10.1371/journal.ppat.1004864.s001
(TIF)

S2 Fig. Flow cytometric analysis of activation marker expression in inhibitor-treated CD4+ T cells.

CD4+ T cells were first pretreated with 10 μM PLD1i, 100nM rapamycin, 10 μM U0126, or 50 μM Myci and then stimulated with anti-CD3/anti-CD28 beads for 48h in the presence or absence of inhibitors. Cells were stained for CD25, CD71, and CD98 expression. Cellular toxicity was determined with LIVE/DEAD Blue viability stain. The frequency and MFI (upper left) of gated populations is indicated in each histogram. Data are representative of two independent experiments.
https://doi.org/10.1371/journal.ppat.1004864.s002
(TIF)

Author Contributions

Conceived and designed the experiments: HET GES TPM WP. Performed the experiments: HET GES TPM AKK. Analyzed the data: HET GES TPM WP. Contributed reagents/materials/analysis tools: HAB CWL RTD. Wrote the paper: HET HAB RTD.

# data[].attributes.attachment_link.title
Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

# data[].attributes.attachment_link.description
Author Summary Replication of all human viruses depends on building blocks derived from the metabolic pathways of the infected host cell. The production of progeny virions requires synthesis of viral nucleic acids from deoxyribonucleotide triphosphates (dNTPs). HIV-1 infection in resting T cells is limited, at least in part, because the levels of critical nucleotides are low.

# data[].attributes.attachment_link.readable_text
Abstract

The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity.

View Full Text

DOI: 10.1126/science.aan8630

# data[].attributes.attachment_link.title
Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

# data[].attributes.attachment_link.description
The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site.