DOT1L/H3K79me2 represses HIV-1 reactivation via recruiting DCAF1.

DOT1L mediates the methylation of histone H3 at lysine 79 and, in turn, the transcriptional activation or repression in a context-dependent manner, yet the regulatory mechanisms and functions of DOT1L/H3K79me remain to be fully explored. Following peptide affinity purification and proteomic analysis, we identified that DCAF1-a component of the E3 ligase complex involved in HIV regulation-is associated with H3K79me2 and DOT1L. Interestingly, blocking the expression or catalytic activity of DOT1L or repressing the expression of DCAF1 significantly enhances the tumor necrosis factor alpha (TNF-α)/nuclear factor κB (NF-κB)-induced reactivation of the latent HIV-1 genome. Mechanistically, upon TNF-α/NF-κB activation, DCAF1 is recruited to the HIV-1 long terminal repeat (LTR) by DOT1L and H3K79me2. Recruited DCAF1 subsequently induces the ubiquitination of NF-κB and restricts its accumulation at the HIV-1 LTR. Altogether, our findings reveal a feedback modulation of HIV reactivation by DOT1L-mediated histone modification regulation and highlight the potential of targeting the DOT1L/DCAF1 axis as a therapeutic strategy for HIV treatment.

KDM5A/B contribute to HIV-1 latent infection and survival of HIV-1 infected cells.

Combinational antiretroviral therapy (cART) suppresses human immunodeficiency virus type 1 (HIV-1) viral replication and pathogenesis in acquired immunodeficiency syndrome (AIDS) patients. However, HIV-1 remains in the latent stage of infection by suppressing viral transcription, which hinders an HIV-1 cure. One approach for an HIV-1 cure is the "shock and kill" strategy. The strategy focuses on reactivating latent HIV-1, inducing the viral cytopathic effect and facilitating the immune clearance for the elimination of latent HIV-1 reservoirs. Here, we reported that the H3K4 trimethylation (H3K4me3)-specific demethylase KDM5A/B play a role in suppressing HIV-1 Tat/LTR-mediated viral transcription in HIV-1 latent cells. Furthermore, we evaluated the potential of KDM5-specific inhibitor JQKD82 as an HIV-1 "shock and kill" agent. Our results showed that JQKD82 increases the H3K4me3 level at HIV-1 5' LTR promoter regions, HIV-1 reactivation, and the cytopathic effects in an HIV-1-latent T cell model. In addition, we identified that the combination of JQKD82 and AZD5582, a non-canonical NF-κB activator, generates a synergistic impact on inducing HIV-1 lytic reactivation and cell death in the T cell. The latency-reversing potency of the JQKD82 and AZD5582 pair was also confirmed in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 aviremic patients and in an HIV-1 latent monocyte. In latently infected microglia (HC69) of the brain, either deletion or inhibition of KDM5A/B results in a reversal of the HIV-1 latency. Overall, we concluded that KDM5A/B function as a host repressor of the HIV-1 lytic reactivation and thus promote the latency and the survival of HIV-1 infected reservoirs.

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.

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes.

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

Schlafen 5 suppresses human immunodeficiency virus type 1 transcription by commandeering cellular epigenetic machinery.

Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family, which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase II to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1-570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.

HIV-1 Tat endocytosis and retention in endolysosomes affects HIV-1 Tat-induced LTR transactivation in astrocytes.

The presence of latent HIV-1 reservoirs in the periphery and brain represents a major obstacle to curing HIV-1 infection. As an essential protein for HIV-1 viral replication, HIV-1 Tat, mostly intracellular, has been implicated in latent HIV-1 infection. From HIV-1 infected cells, HIV-1 Tat is actively secreted and bystander cells uptake the released Tat whereupon it is endocytosed and internalized into endolysosomes. However, to activate the HIV-1 LTR promoter and increase HIV-1 replication, HIV-1 Tat must first escape from the endolysosomes and then enter the nucleus. Here, we tested the hypothesis that HIV-1 Tat can accumulate in endolysosomes and contribute to the activation of latent HIV-1 in astrocytes. Using U87MG astrocytoma cells expressing HIV-1 LTR-driven luciferase and primary human astrocytes we found that exogenous HIV-1 Tat enters endolysosomes, resides in endolysosomes for extended periods of time, and induces endolysosome de-acidification as well as enlargement. The weak base chloroquine promoted the release of HIV-1 Tat from endolysosomes and induced HIV-1 LTR transactivation. Similar results were observed by activating endolysosome Toll-like receptor 3 (TLR3) and TLR7/8. Conversely, pharmacological block of TLRs and knocking down expression levels of TLR3 and TLR7, but not TLR8, prevented endolysosome leakage and attenuated HIV-1 Tat-mediated HIV-1 LTR transactivation. Our findings suggest that HIV-1 Tat accumulation in endolysosomes may play an important role in controlling HIV-1 transactivation.

UHRF1 Suppresses HIV-1 Transcription and Promotes HIV-1 Latency by Competing with p-TEFb for Ubiquitination-Proteasomal Degradation of Tat.

HIV-1 remains incurable due to viral reservoirs, which lead to durably latent HIV infection. Identifying novel host factors and deciphering the molecular mechanisms involved in the establishment and maintenance of latency are critical to discover new targets for the development of novel anti-HIV agents. Here, we show that ubiquitin-like with PHD and RING finger domain 1 (UHRF1) modulates HIV-1 5'-long terminal repeat (LTR)-driven transcription of the viral genome as a novel HIV-1 restriction factor. Correspondingly, UHRF1 depletion reversed the latency of HIV-1 proviruses. Mechanistically, UHRF1 competed with positive transcription factor b (p-TEFb) for the binding to the cysteine-rich motifs of HIV-1 Tat via its TTD, PHD, and RING finger domains. Furthermore, UHRF1 mediated K48-linked ubiquitination and proteasomal degradation of Tat in RING-dependent ways, leading to the disruption of Tat/cyclin T1/CDK9 complex and consequential impediment of transcription elongation. In summary, our findings revealed that UHRF1 is an important mediator of HIV-1 latency by controlling Tat-mediated transcriptional activation, providing novel insights on host-pathogen interaction for modulating HIV-1 latency, beneficial for the development of anti-AIDS therapies. IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. In our work, we identified a critical role of host factor ubiquitin-like with PHD and RING finger domain 1 (UHRF1) in HIV-1 latency via the modulation of the viral protein Tat stability. By disrupting the Tat/cyclin T1/CDK9 complex, UHRF1 promotes the suppression of HIV-1 transcription and maintenance of HIV-1 latency. Our findings provide novel insights in controlling Tat expression via host-pathogen interaction for modulating HIV-1 latency. Based on our results, modulating UHRF1 expression or activity by specific inhibitors is a potential therapeutic strategy for latency reversal in HIV-1 patients.

Differences in Transcriptional Dynamics Between T-cells and Macrophages as Determined by a Three-State Mathematical Model.

HIV-1 viral transcription persists in patients despite antiretroviral treatment, potentially due to intermittent HIV-1 LTR activation. While several mathematical models have been explored in the context of LTR-protein interactions, in this work for the first time HIV-1 LTR model featuring repressed, intermediate, and activated LTR states is integrated with generation of long (env) and short (TAR) RNAs and proteins (Tat, Pr55, and p24) in T-cells and macrophages using both cell lines and infected primary cells. This type of extended modeling framework allows us to compare and contrast behavior of these two cell types. We demonstrate that they exhibit unique LTR dynamics, which ultimately results in differences in the magnitude of viral products generated. One of the distinctive features of this work is that it relies on experimental data in reaction rate computations. Two RNA transcription rates from the activated promoter states are fit by comparison of experimental data to model predictions. Fitting to the data also provides estimates for the degradation/exit rates for long and short viral RNA. Our experimentally generated data is in reasonable agreement for the T-cell as well macrophage population and gives strong evidence in support of using the proposed integrated modeling paradigm. Sensitivity analysis performed using Latin hypercube sampling method confirms robustness of the model with respect to small parameter perturbations. Finally, incorporation of a transcription inhibitor (F07#13) into the governing equations demonstrates how the model can be used to assess drug efficacy. Collectively, our model indicates transcriptional differences between latently HIV-1 infected T-cells and macrophages and provides a novel platform to study various transcriptional dynamics leading to latency or activation in numerous cell types and physiological conditions.

The dual-target approach in viral HIV-1 viremia testing: An added value to virological monitoring?

New methods of HIV-1 RNA quantification based on dual-target detection are increasingly used in HIV viral load monitoring, but clinical implications and impact of dual-target detection on HIV-1 infection management are not established. Aptima HIV-1 Quant Dx assay is a last generation HIV viral load method, that uses pol and LTR as simultaneous target, providing quantitative results based mainly on pol target, while LTR target is used to report the results when pol signal is absent. In our laboratory, about 6% of results of all HIV-1 viral load tests performed with this platform in one year period resulted from LTR signal. Interestingly, LTR-based viremia (sometimes exceeding 1,000 copies/mL) was observed in a small proportion (up to 1%) of patients under ART, considered for long time virologically suppressed on the basis of a single target (pol-based) assay. Male gender, >700 vs <200 CD4 cell/mL and dual therapy including NRTI plus either NNRTI, or PI/b or INSTI were independently associated with increased risk of LTR-based HIV-1 viral load detection by multivariable logistic regression. A significant linear correlation was observed between LTR-based HIV-1 RNA levels and PBMC-associated proviral DNA. Moreover, in a small group of patients with HIV-1 RNA levels >200 copies/mL, longitudinal assessments showed parallel kinetics between plasma viremia and proviral DNA. Sequencing of pol region for drug resistance assessment in patients with LTR-based viremia failed on plasma HIV-1 RNA, while it was successful on proviral DNA. The detection/quantification of HIV-1 viremia based only on LTR signal with a dual target assay in samples resulting undetectable with the more conventional target pol needs accurate evaluation; unravelling the biological basis of this phenomenon, here described for the first time, is mandatory to establish relevance and implication by both pathogenetic (i.e. infectivity of LTR-detected viruses, reservoir turnover, immune activation, etc.) and clinical standpoint.

Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR.

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

Increased expression of BCL11B and its recruited chromatin remodeling factors during highly active antiretroviral therapy synergistically represses the transcription of human immunodeficiency virus type 1 and is associated with residual immune activation.

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.

Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1 replication by displacing the polycomb repressive complex 2 from binding to the LTR promoter.

Long noncoding RNAs (lncRNAs) may either repress or activate HIV-1 replication and latency; however, specific mechanisms for their action are not always clear. In HIV-1 infected CD4+ T cells, we performed RNA-Sequencing (RNA-Seq) analysis and discovered an up-regulation of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), an lncRNA previously described in cancer cells that associate with cancer pathogenesis. Moreover, we found that MALAT1 promoted HIV-1 transcription and infection, as its knockdown by CRISPR/Cas9 markedly reduced the HIV-1 long terminal repeat (LTR)-driven gene transcription and viral replication. Mechanistically, through an association with chromatin modulator polycomb repressive complex 2 (PRC2), MALAT1 detached the core component enhancer of zeste homolog 2 (EZH2) from binding with HIV-1 LTR promoter, and thus removed PRC2 complex-mediated methylation of histone H3 on lysine 27 (H3K27me3) and relieved epigenetic silencing of HIV-1 transcription. Moreover, the reactivation of HIV-1 stimulated with latency reversal agents (LRAs) induced MALAT1 expression in latently infected cells. Successful combination antiretroviral therapy (cART) was accompanied by significantly diminished MALAT1 expression in patients, suggesting a positive correlation of MALAT1 expression with HIV-1 replication. Our data have identified MALAT1 as a promoter of HIV-1 transcription, and suggested that MALAT1 may be targeted for the development of new therapeutics.

Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.

HIV 2-LTR experiment design optimization.

Clinical trials are necessary in order to develop treatments for diseases; however, they can often be costly, time consuming, and demanding to the patients. This paper summarizes several common methods used for optimal design that can be used to address these issues. In addition, we introduce a novel method for optimizing experiment designs applied to HIV 2-LTR clinical trials. Our method employs Bayesian techniques to optimize the experiment outcome by maximizing the Expected Kullback-Leibler Divergence (EKLD) between the a priori knowledge of system parameters before the experiment and the a posteriori knowledge of the system parameters after the experiment. We show that our method is robust and performs equally well if not better than traditional optimal experiment design techniques.

Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA.

People living with HIV/AIDS on antiretroviral therapy have increased risk of non-AIDS-defining cancers (NADCs). However, the underlying mechanism for development and progression of certain NADCs remains obscure. Here we show that exosomes released from HIV-infected T cells and those purified from blood of HIV-positive patients stimulate proliferation, migration and invasion of oral/oropharyngeal and lung cancer cells. The HIV transactivation response (TAR) element RNA in HIV-infected T-cell exosomes is responsible for promoting cancer cell proliferation and inducing expression of proto-oncogenes and Toll-like receptor 3 (TLR3)-inducible genes. These effects depend on the loop/bulge region of the molecule. HIV-infected T-cell exosomes rapidly enter recipient cells through epidermal growth factor receptor (EGFR) and stimulate ERK1/2 phosphorylation via the EGFR/TLR3 axis. Thus, our findings indicate that TAR RNA-containing exosomes from HIV-infected T cells promote growth and progression of particular NADCs through activation of the ERK cascade in an EGFR/TLR3-dependent manner.

Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR.

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an essential role in HIV-1 gene transcription. Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression through direct interactions with the transactivation-responsive region (TAR) element and other cis elements in the LTR. The TAR-independent Tat-mediated LTR transactivation is modulated by several host factors, but the mechanism is not fully understood. RESULTS: Here, we report that Tat interacts with the Rel homology domain of RelB through its core region. Furthermore, RelB significantly increases Tat-mediated transcription of the HIV-1 LTR and viral gene expression, which is independent of the TAR. Both Tat and RelB are recruited to the HIV-1 promoter, of which RelB facilitates the recruitment of Tat to the viral LTR. The NF-κB elements are key to the accumulation of Tat and RelB on the LTR. Knockout of RelB reduces the accumulation of RNA polymerase II on the LTR, and decreases HIV-1 gene transcription. Together, our data suggest that RelB contributes to HIV-1 transactivation. CONCLUSIONS: Our results demonstrate that RelB interacts with Tat and enhances TAR-independent activation of HIV-1 LTR promoter, which adds new insights into the multi-layered mechanisms of Tat in regulating the gene expression of HIV-1.

Modulated expression of the HIV-1 2LTR zinc finger efficiently interferes with the HIV integration process.

Lentiviral vectors have emerged as the most efficient system to stably transfer and insert genes into cells. By adding a tetracycline (Tet)-inducible promoter, transgene expression delivered by a lentiviral vector can be expressed whenever needed and halted when necessary. Here we have constructed a doxycycline (Dox)-inducible lentiviral vector which efficiently introduces a designed zinc finger protein, 2-long terminal repeat zinc-finger protein (2LTRZFP), into hematopoietic cell lines and evaluated its expression in pluripotent stem cells. As a result this lentiviral inducible system can regulate 2LTRZFP expression in the SupT1 T-cell line and in pluripotent stem cells. Using this vector, no basal expression was detected in the T-cell line and its induction was achieved with low Dox concentrations. Remarkably, the intracellular regulatory expression of 2LTRZFP significantly inhibited HIV-1 integration and replication in HIV-inoculated SupT1 cells. This approach could provide a potential tool for gene therapy applications, which efficiently control and reduce the side effect of therapeutic genes expression.