HIV-1 Transcription Research Articles

Targeting Viral Transcription for HIV Cure Strategies.

Combination antiretroviral therapy (ART) suppresses viral replication to undetectable levels, reduces mortality and morbidity, and improves the quality of life of people living with HIV (PWH). However, ART cannot cure HIV infection because it is unable to eliminate latently infected cells. HIV latency may be regulated by different HIV transcription mechanisms, such as blocks to initiation, elongation, and post-transcriptional processes. Several latency-reversing (LRA) and -promoting agents (LPA) have been investigated in clinical trials aiming to eliminate or reduce the HIV reservoir. However, none of these trials has shown a conclusive impact on the HIV reservoir. Here, we review the cellular and viral factors that regulate HIV-1 transcription, the potential pharmacological targets and genetic and epigenetic editing techniques that have been or might be evaluated to disrupt HIV-1 latency, the role of miRNA in post-transcriptional regulation of HIV-1, and the differences between the mechanisms regulating HIV-1 and HIV-2 expression.

Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner.

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that, in a variety of cell types, including primary immune cells, SLFN14 regulates the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibits the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal RNA degradation is involved. Therefore, we propose that SLFN14 impairs the expression of HIV-1 transcripts rich in rare codons, in a catalytic-dependent manner.

Dynamic modulation of the non-canonical NF-κB signaling pathway for HIV shock and kill.

HIV cure still remains an elusive target. The "Shock and Kill" strategy which aims to reactivate HIV from latently infected cells and subsequently kill them through virally induced apoptosis or immune mediated clearance, is the subject of widespread investigation. NF-κB is a ubiquitous transcription factor which serves as a point of confluence for a number of intracellular signaling pathways and is also a crucial regulator of HIV transcription. Due to its relatively lower side effect profile and proven role in HIV transcription, the non-canonical NF-κB pathway has emerged as an attractive target for HIV reactivation, as a first step towards eradication. A comprehensive review examining this pathway in the setting of HIV and its potential utility to cure efforts is currently lacking. This review aims to summarize non-canonical NF-κB signaling and the importance of this pathway in HIV shock-and-kill efforts.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies.

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.

Gesicles packaging dCas9-VPR ribonucleoprotein complexes can combine with vorinostat and promote HIV proviral transcription

Despite the success of combination antiretroviral therapies (cART) in HIV treatment, a cure for HIV remains elusive. Scientists postulate that HIV latent reservoirs may be a vital target in curative strategies. Vorinostat is a latency-reversing agent which has demonstrated some effectiveness in reactivating latent HIV, but complementary therapies may be essential to enhance its efficacy. One such approach may utilize the CRISPR/Cas9 system which has evolved to include transcriptional activators such as dCas9-VPR. In this study, we explored the effects of combining vorinostat coupled with gesicle-mediated delivery of dCas9-VPR in promoting the transcription of integrated HIV proviruses in HIV-NanoLuc CHME-5 microglia and J-Lat 10.6 lymphocytes. We confirmed that dCas9-VPR ribonucleoprotein complexes can be packaged into gesicles and application to cells successfully induced HIV transcription through interactions with the HIV LTR. Vorinostat also induced significant increases in proviral transcription but generated inhibition of cellular proliferation (microglia) or cell viability (lymphocytes) starting at 1000nM and higher concentrations. Experiments combining dCas9-VPR gesicles and vorinostat confirmed the enhanced transcriptional activation of the HIV provirus in microglia but not lymphocytes. Thus, a combination of dCas9-VPR gesicles with other latency reversing agents may provide a complementary method to activate latent HIV in future studies utilizing patient-derived cells or small animal models.

Enhanced NF-κB activation via HIV-1 Tat-TRAF6 cross-talk.

The Tat proteins of HIV-1 and simian immunodeficiency virus (SIV) are essential for activating viral transcription. In addition, Tat stimulates nuclear factor κB (NF-κB) signaling pathways to regulate viral gene expression although its molecular mechanism is unclear. Here, we report that Tat directly activates NF-κB through the interaction with TRAF6, which is an essential upstream signaling molecule of the canonical NF-κB pathway. This interaction increases TRAF6 oligomerization and auto-ubiquitination, as well as the synthesis of K63-linked polyubiquitin chains to further activate the NF-κB pathway and HIV-1 transcription. Moreover, ectopic expression of TRAF6 significantly activates HIV-1 transcription, whereas TRAF6 knockdown inhibits transcription. Furthermore, Tat-mediated activation of NF-κB through TRAF6 is conserved among HIV-1, HIV-2, and SIV isolates. Our study uncovers yet another mechanism by which HIV-1 subverts host transcriptional pathways to enhance its own transcription.

Functional Analysis of KAP1/TRIM28 Requirements for HIV-1 Transcription Activation.

HIV-1 latency maintenance and reactivation are regulated by several viral and host factors. One such factor is Krüppel-associated box (KRAB)-associated protein 1 (KAP1: also named TRIM28 or TIF1β). While initial studies have revealed KAP1 to be a positive regulator of latency reversal in transformed and primary CD4+ T cells, subsequent studies have proposed KAP1 to be a repressor required for latency maintenance. Given this discrepancy, in this study, we re-examine KAP1 transcription regulatory functions using a chemical genetics strategy to acutely deplete KAP1 expression to avoid the accumulation of indirect effects. Notably, KAP1 acute loss partially decreased HIV-1 promoter activity in response to activating signals, a function that can be restored upon complementation with exogenous KAP1, thus revealing that KAP1-mediated activation is on target. By combining comprehensive KAP1 domain deletion and mutagenesis in a cell-based reporter assay, we genetically defined the RING finger domain and an Intrinsically Disordered Region as key activating features. Together, our study solidifies the notion that KAP1 activates HIV-1 transcription by exploiting its multi-domain protein arrangement via previously unknown domains and functions.

Metformin Treatment Leads to Increased HIV Transcription and Gene Expression through Increased CREB Phosphorylation and Recruitment to the HIV LTR Promoter.

Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with the long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB phosphorylation/activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production through increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using Metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV.

Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

RNA-FISH for HIV Transcription/Localization Analysis.

RNA fluorescence in situ hybridization (FISH) serves as a method for visualizing specific RNA molecules within cells. Its primary utility lies in the observation of messenger RNA (mRNA) molecules associated with particular genes of significance. This technique can also be applied to examine viral transcription and the localization of said transcripts within infected cells. In this context, we provide a comprehensive protocol for the detection, localization, and quantification of HIV-1 transcripts in mammalian cell lines. This encompasses the preparation of required reagents, cellular treatments, visualization, and the subsequent analysis of the data acquired. These parameters play a pivotal role in enhancing our comprehension of the molecular processes during infection, particularly at the crucial transcription phase of the viral life cycle.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT− mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy

The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

HIV-1 Transcription Inhibition Using Small RNA-Binding Molecules.

The HIV-1 transactivator protein Tat interacts with the transactivation response element (TAR) at the three-nucleotide UCU bulge to facilitate the recruitment of transcription elongation factor-b (P-TEFb) and induce the transcription of the integrated proviral genome. Therefore, the Tat-TAR interaction, unique to the virus, is a promising target for developing antiviral therapeutics. Currently, there are no FDA-approved drugs against HIV-1 transcription, suggesting the need to develop novel inhibitors that specifically target HIV-1 transcription. We have identified potential candidates that effectively inhibit viral transcription in myeloid and T cells without apparent toxicity. Among these candidates, two molecules showed inhibition of viral protein expression. A molecular docking and simulation approach was used to determine the binding dynamics of these small molecules on TAR RNA in the presence of the P-TEFb complex, which was further validated by a biotinylated RNA pulldown assay. Furthermore, we examined the effect of these molecules on transcription factors, including the SWI/SNF complex (BAF or PBAF), which plays an important role in chromatin remodeling near the transcription start site and hence regulates virus transcription. The top candidates showed significant viral transcription inhibition in primary cells infected with HIV-1 (98.6). Collectively, our study identified potential transcription inhibitors that can potentially complement existing cART drugs to address the current therapeutic gap in current regimens. Additionally, shifting of the TAR RNA loop towards Cyclin T1 upon molecule binding during molecular simulation studies suggested that targeting the TAR loop and Tat-binding UCU bulge together should be an essential feature of TAR-binding molecules/inhibitors to achieve complete viral transcription inhibition.

An RNA excited conformational state at atomic resolution

Sparse and short-lived excited RNA conformational states are essential players in cell physiology, disease, and therapeutic development, yet determining their 3D structures remains challenging. Combining mutagenesis, NMR spectroscopy, and computational modeling, we determined the 3D structural ensemble formed by a short-lived (lifetime ~2.1 ms) lowly-populated (~0.4%) conformational state in HIV-1 TAR RNA. Through a strand register shift, the excited conformational state completely remodels the 3D structure of the ground state (RMSD from the ground state = 7.2 ± 0.9 Å), forming a surprisingly more ordered conformational ensemble rich in non-canonical mismatches. The structure impedes the formation of the motifs recognized by Tat and the super elongation complex, explaining why this alternative TAR conformation cannot activate HIV-1 transcription. The ability to determine the 3D structures of fleeting RNA states using the presented methodology holds great promise for our understanding of RNA biology, disease mechanisms, and the development of RNA-targeting therapeutics.

Breaking the Silence: Regulation of HIV Transcription and Latency on the Road to a Cure.

Antiretroviral therapy (ART) has brought the HIV/AIDS epidemic under control, but a curative strategy for viral eradication is still needed. The cessation of ART results in rapid viral rebound from latently infected CD4+ T cells, showing that control of viral replication alone does not fully restore immune function, nor does it eradicate viral reservoirs. With a better understanding of factors and mechanisms that promote viral latency, current approaches are primarily focused on the permanent silencing of latently infected cells ("block and lock") or reactivating HIV-1 gene expression in latently infected cells, in combination with immune restoration strategies to eliminate HIV infected cells from the host ("shock and kill"). In this review, we provide a summary of the current, most promising approaches for HIV-1 cure strategies, including an analysis of both latency-promoting agents (LPA) and latency-reversing agents (LRA) that have shown promise in vitro, ex vivo, and in human clinical trials to reduce the HIV-1 reservoir.

Emerging role of cyclophilin A in HIV-1 infection: from producer cell to the target cell nucleus.

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.

HIV-1 Restriction in Systemic Lupus Erythematosus Involves Iron Homeostasis

INTRODUCTION: Over the past two decades, there has been a growing emphasis on host restriction factors. However, a comprehensive understanding of their role in modulating HIV-1 infection in vivo requires further detailed investigation. Intracellular iron levels can affect HIV-1 replication by influencing expression of host cell viral restriction factors. Systemic Lupus Erythematosus (SLE) is characterized by upregulation of interferon response and the coexistence of SLE and HIV infection is rare. In lupus nephritis, deregulation of proteins involved in iron homeostasis have also been identified, with hepcidin suggested to play a role in disease severity . Hif1α, a protein regulated by hypoxia and under iron depletion, and reported to block HIV transcription, is upregulated in SLE. High levels of IL-10 and IL-16 which inhibits HIV infection are upregulated in SLE. APOBECs and oxysterols and Interferon Regulating Factors (IRFs) are also known to be upregulated in SLE. The current study is aimed to investigate ex vivo HIV-1 infection in peripheral blood mononuclear cells (PBMCs) derived from individuals SLE and identify factors that contributed to this inhibition. METHODS: We utilized replication deficient VSVg-pseudotype HIV-1 and replication competent HIV-IIIB to infect SLE PBMCs. Real-time PCR was conducted to determine the upregulation of genes involved in HIV-1 replication. RNA-seq of PBMCs were done to identify altered restriction factors. RESULTS: We investigated one round of HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from individuals with Systemic Lupus Erythematosus (SLE) and compared them to control PBMCs. We found that the HIV-1 infection in SLE-derived PBMCs was significantly reduced, as indicated by lower mRNA levels of Gag and Nef, two essential viral proteins. Our ongoing study, which focused on the inhibition of HIV-1 replication ex vivo in Sickle Cell Disease (SCD), revealed interesting findings. RNA sequencing of PBMCs from individuals with SCD showed an upregulation of APOBEC3A, APOBEC3B, and CH25H, which are known restriction factors against HIV-1. Given these intriguing results, we decided to analyze the RNA expression of these restriction factors in SLE-derived PBMCs. Surprisingly, we found a significant increase in the mRNA levels of APOBEC3A, APOBEC3B, and CH25H in SLE-derived PBMCs as well. This suggests that the enhanced expression of these restriction factors could be contributing to the reduced HIV-1 infection observed in SLE-derived PBMCs. Additionally, we explored the RNA expression of HIF-1α and TFR1, which are iron-regulated factors, and found that their mRNA levels were upregulated in SLE-derived PBMCs. Furthermore, we observed an upregulation of interferon regulatory factors, including IL-10, IFN-β, and IRF-7, which are involved in immune responses and antiviral defense mechanisms. Interestingly IRF-7 was upregulated in SCD PBMCs too. DISSUCION: The ongoing study in our lab on SCD and the study reported here are known for deregulated iron metabolism. Interestingly on one hand PBMCs derived from both inhibit HIV-1 and on other have some common restriction factors, iron regulated genes and IRF upregulated. Iron-regulated factors, such as TFR1, HIF-1α and ferroportin have been associated with antiviral effects. Modulating the expression or activity of these factors could influence HIV infection. CONCLUSIONs: Taken together, our findings indicate that SLE-derived PBMCs exhibit a distinct gene expression profile that may confer protection against HIV-1 infection. The upregulation of APOBEC3A, APOBEC3B, CH25H, HIF-1α, TFR1, and interferon regulatory factors could collectively contribute to the observed reduction in HIV-1 replication in SLE-derived PBMCs. Further investigation is needed to fully understand the mechanisms underlying these observations and their implications for HIV-1 pathogenesis under altered intracellular iron levels influencing HIV-1 replication by modulating host cell factors. Acknowledgments This work was supported by NIH Research Grants 1R01HL125005, 5U54MD007597, P30AI087714 and P50AI150476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

HIV-2 inhibits HIV-1 gene expression via two independent mechanisms during cellular co-infection.

Twenty-five years after the first report that HIV-2 infection can reduce HIV-1-associated pathogenesis in dual-infected patients, the mechanisms are still not well understood. We explored these mechanisms in cell culture and showed first that these viruses can co-infect individual cells. Under specific conditions, HIV-2 inhibits HIV-1 through two distinct mechanisms, a broad-spectrum interferon response and an HIV-1-specific inhibition conferred by the HIV-2 TAR. The former could play a prominent role in dually infected individuals, whereas the latter targets HIV-1 promoter activity through competition for HIV-1 Tat binding when the same target cell is dually infected. That mechanism suppresses HIV-1 transcription by stalling RNA polymerase II complexes at the promoter through a minimal inhibitory region within the HIV-2 TAR. This work delineates the sequence of appearance and the modus operandi of each mechanism.

Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.