HIV-1 Proviral Transcription Research Articles

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.

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.

Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency.

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compound's ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.

Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation.

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the bivalent promoter in the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. In an attempt to suppress latent HIV-1 in a stable fashion, we knocked down the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and was associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and also induced DNA methylation at specific sites in the 5'LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. DNA methylation was also rapidly lost after removal of drug, suggesting active and rapid DNA-demethylation of the HIV LTR. Thus, induction of epigenetic silencing by histone and DNA methylation appears to be insufficient to permanently silence HIV-1 proviral transcription.

Cleavage and Polyadenylation Specificity Factor 6 Is Required for Efficient HIV-1 Latency Reversal.

ABSTRACTThe HIV-1 latent reservoir is the major barrier to an HIV cure. Due to low levels or lack of transcriptional activity, HIV-1 latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies based on protein expression. To target the latent reservoir, further understanding of HIV-1 proviral transcription is required. In this study, we demonstrate a novel role for cleavage and polyadenylation specificity factor 6 (CPSF6) in HIV-1 transcription. We show that knockout of CPSF6 hinders reactivation of latent HIV-1 proviruses by PMA in primary CD4+ cells. CPSF6 knockout reduced HIV-1 transcription, concomitant with a drastic reduction in the phosphorylation levels of Pol II and CDK9. Knockout of CPSF6 led to abnormal stabilization of protein phosphatase 2A (PP2A) subunit A, which then acted to dephosphorylate CDK9, downmodulating CDK9's ability to phosphorylate the Pol II carboxy-terminal domain. In agreement with this mechanism, incubation with the PP2A inhibitor, LB100, restored HIV-1 transcription in the CPSF6 knockout cells. Destabilization of PP2A subunit A occurs in the ubiquitin proteasome pathway, wherein CPSF6 acts as a substrate adaptor for the ITCH ubiquitin ligase. Our observations reveal a novel role of CPSF6 in HIV-1 transcription, which appears to be independent of its known roles in cleavage and polyadenylation and the targeting of preintegration complexes to the chromatin for viral DNA integration.

HIV-1 Proviral Transcription and Latency in the New Era.

Three decades of extensive work in the HIV field have revealed key viral and host cell factors controlling proviral transcription. Various models of transcriptional regulation have emerged based on the collective information from in vitro assays and work in both immortalized and primary cell-based models. Here, we provide a recount of the past and current literature, highlight key regulatory aspects, and further describe potential limitations of previous studies. We particularly delve into critical steps of HIV gene expression including the role of the integration site, nucleosome positioning and epigenomics, and the transition from initiation to pausing and pause release. We also discuss open questions in the field concerning the generality of previous regulatory models to the control of HIV transcription in patients under suppressive therapy, including the role of the heterogeneous integration landscape, clonal expansion, and bottlenecks to eradicate viral persistence. Finally, we propose that building upon previous discoveries and improved or yet-to-be discovered technologies will unravel molecular mechanisms of latency establishment and reactivation in a “new era”.

Structural and functional insight into the effect of AFF4 dimerization on activation of HIV-1 proviral transcription

Super elongation complex (SEC) is a positive regulator of RNA polymerase II, which is required for HIV-1 proviral transcription. AFF1/4 is the scaffold protein that recruits other components of SEC and forms dimer depending on its THD domain (TPRL with Handle Region Dimerization Domain). Here we report the crystal structure of the human AFF4-THD at the resolution of 2.4 Å. The α4, α5, and α6 of one AFF4-THD mediate the formation of a dimer and pack tightly against the equivalent part of the second molecule in the dimer of AFF-THD. Mutagenesis analysis revealed that single mutations of either Phe1014 or Tyr1096 of AFF4 to alanine impair the formation of the AFF4 dimer. In addition, transactivation assay also indicated that Phe1014 and Tyr1096 of AFF4 are critical to the transactivation activity of AFF4. Interestingly, the corresponding residues Phe1063 and Tyr1145 in AFF1 have an effect on the transactivation of HIV-1 provirus. However, such mutations of AFF1/4 have no effect on the interaction of AFF1/4 with other subunits of the SEC. Together, our data demonstrated that the dimerization of AFF1/4 is essential to transactivation of HIV-1 provirus.

PP 6.8.7 - CD4 T cells from patients with chronic myeloid leukemia are resistant to HIV-1 proviral integration and transcription after prolonged withdrawal of treatment with tyrosine kinase inhibitors

PP 6.8.7 - CD4 T cells from patients with chronic myeloid leukemia are resistant to HIV-1 proviral integration and transcription after prolonged withdrawal of treatment with tyrosine kinase inhibitors

HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

BackgroundHIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality.ResultsMass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR—Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E—poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue.ConclusionPhosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.

P-D2 TRIM22 binds to CIITA and sequesters it into nuclear bodies containing TRIM19/PML and Cyclin T1: Implications for HIV-1 infection

Host restriction factors represent a defence mechanism of the innate immune system against viral pathogens, such as HIV-1. Many studies focused on the interplay between restriction factors and HIV-1 have greatly improved our knowledge about the biology of the virus and the response of the host, but to date there is no evidence on of a possible interaction between host restriction factors to counteract the virus. Here we show that TRIM22 and CIITA, 2 cellular proteins previously shown by us to inhibit HIV-1 proviral transcription with different mechanisms, interact in vivo and co-localize in nuclear bodies whose formation is hierarchically controlled by TRIM22. Importantly, TRIM19/Promyelocytic Leukemia (PML) protein, another repressor of HIV-1 transcription also acting before proviral integration, co-localized in these nuclear bodies upon TRIM22 expression induced by IFN-γ. Finally, TRIM22 nuclear bodies also contained CyclinTI, a crucial elongation factor of HIV-1 primary transcripts. These findings show that TRIM22 nuclear bodies are a site of recruitment of factors crucial for the regulation of HIV-1 transcription and highlight the potential existence of a concerted action between TRIM22, CIITA and TRIM19/PML to maintain a state of proviral latency at least in myeloid cells.

BCA2/Rabring7 Interferes with HIV-1 Proviral Transcription by Enhancing the SUMOylation of IκBα.

ABSTRACTBCA2/Rabring7 is a BST2 cofactor that promotes the lysosomal degradation of trapped HIV-1 virions but also functions as a BST2-independent anti-HIV factor by targeting Gag for lysosomal degradation. Since many antiviral factors regulate the NF-κB innate signaling pathway, we investigated whether BCA2 is also connected to this proinflammatory cascade. Here, we show for the first time that BCA2 is induced by NF-κB-activating proinflammatory cytokines and that upregulation of BCA2 provides regulatory negative feedback on NF-κB. Specifically, BCA2 serves as an E3 SUMO ligase in the SUMOylation of IκBα, which in turn enhances the sequestration of NF-κB components in the cytoplasm. Since HIV-1 utilizes NF-κB to promote proviral transcription, the BCA2-mediated inhibition of NF-κB significantly decreases the transcriptional activity of HIV-1 (up to 4.4-fold in CD4+ T cells). Therefore, our findings indicate that BCA2 poses an additional barrier to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, it also significantly restricts HIV-1 transcription by inhibiting the NF-κB pathway.IMPORTANCE Understanding the interactions between HIV-1 and its host cells is highly relevant to the design of new drugs aimed at eliminating HIV-1 from infected individuals. We have previously shown that BCA2, a cofactor of BST2 in the restriction of HIV-1, also prevents virion assembly in a BST2-independent manner. In this study, we found that BCA2 negatively regulates the NF-κB pathway—a signaling cascade necessary for HIV-1 replication and infectivity—which in turn detrimentally affects proviral transcription and virus propagation. Thus, our results indicate that, besides its previously described functions as an antiviral factor, BCA2 poses an additional barrier to HIV-1 replication at the transcriptional level.

Structural basis for ELL2 and AFF4 activation of HIV-1 proviral transcription

The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the super elongation complex required for HIV-1 proviral transcription. Here we report the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4–ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.

Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex.

HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.

Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication.

A rare subset of HIV-1-infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1-infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1-infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection.

Blimp-1, an intrinsic factor that represses HIV-1 proviral transcription in memory CD4+ T cells.

CD4(+) T cell subsets differentially support HIV-1 replication. For example, quiescent CD4(+) memory T cells are susceptible to HIV-1 infection but do not support robust HIV-1 transcription and have been implicated as the primary reservoir of latent HIV-1. T cell transcription factors that regulate maturation potentially limit HIV-1 transcription and mediate the establishment and maintenance of HIV-1 latency. We report that B lymphocyte-induced maturation protein-1 (Blimp-1), a critical regulator of B and T cell differentiation, is highly expressed in memory CD4(+) T cells compared with naive CD4(+) T cells and represses basal and Tat-mediated HIV-1 transcription. Blimp-1 binds an IFN-stimulated response element within HIV-1 provirus, and it is displaced following T cell activation. Reduction of Blimp-1 in infected primary T cells including CD4(+) memory T cells increases RNA polymerase II processivity, histone acetylation, and baseline HIV-1 transcription. Therefore, the transcriptional repressor, Blimp-1, is an intrinsic factor that predisposes CD4(+) memory T cells to latent HIV-1 infection.

Phosphatase PPM1A negatively regulates P-TEFb function in resting CD4+ T cells and inhibits HIV-1 gene expression

BackgroundProcessive elongation of the integrated HIV-1 provirus is dependent on recruitment of P-TEFb by the viral Tat protein to the viral TAR RNA element. P-TEFb kinase activity requires phosphorylation of Thr186 in the T-loop of the CDK9 subunit. In resting CD4+T cells, low levels of T-loop phosphorylated CDK9 are found, which increase significantly upon activation. This suggests that the phosphorylation status of the T-loop is actively regulated through the concerted actions of cellular proteins such as Ser/Thr phosphatases. We investigated the role of phosphatase PPM1A in regulating CDK9 T-loop phosphorylation and its effect on HIV-1 proviral transcription.ResultsWe found that overexpression of PPM1A inhibits HIV-1 gene expression during viral infection and this required PPM1A catalytic function. Using an artificial CDK tethering system, we further found that PPM1A inhibits CDK9, but not CDK8 mediated activation of the HIV-1 LTR. SiRNA depletion of PPM1A in resting CD4+T cells increased the level of CDK9 T-loop phosphorylation and enhanced HIV-1 gene expression. We also observed that PPM1A protein levels are relatively high in resting CD4+T cells and are not up-regulated upon T cell activation.ConclusionsOur results establish a functional link between HIV-1 replication and modulation of CDK9 T-loop phosphorylation by PPM1A. PPM1A represses HIV-1 gene expression by inhibiting CDK9 T-loop phosphorylation, thus reducing the amount of active P-TEFb available for recruitment to the viral LTR. We also infer that PPM1A enzymatic activity in resting and activated CD4+ T cells are likely regulated by as yet undefined factors.

RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Inhibits HIV-1 Transcription in Monocytes/Macrophages and Is Decreased in Brain Tissue from Patients with AIDS

Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-kappaB to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.

CD28-dependent HIV-1 Transcription Is Associated with Vav, Rac, and NF-κB Activation

Activation of HIV-1-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; however, the mechanism behind this enhanced virus expression is unknown. Jurkat T cells and primary CD4+ T cells expressing a CD8 alpha/CD28 chimeric receptor containing a mutation at tyrosine 200 in the cytoplasmic tail were unable to fully induce HIV-1 proviral transcription in response to CD8 alpha/28 receptor cross-linking in comparison to CD28 costimulation. The loss of transactivation seen with the mutant chimeric receptor correlated with a decrease in Vav tyrosine phosphorylation. CD28-dependent activation of HIV-1 transcription also required the GTPase activity of Rac1, which was not activated during costimulation with the mutated receptor. Furthermore, the mutated receptor was unable to induce NF-kappa B DNA binding or transactivation, as demonstrated by electromobility shift assays and HIV-1 long terminal repeat and NF-kappa B-dependent reporter constructs. These studies show that signaling events initiated by tyrosine 200 of CD28 are required for efficient expression of HIV-1 transcription in activated T cells.