Human Immunodeficiency Virus Type 1 Transcription Research Articles

Post-Transcriptional HIV-1 Latency: A Promising Target for Therapy?

Human Immunodeficiency Virus type 1 (HIV-1) latency represents a significant hurdle in finding a cure for HIV-1 infections, despite tireless research efforts. This challenge is partly attributed to the intricate nature of HIV-1 latency, wherein various host and viral factors participate in multiple physiological processes. While substantial progress has been made in discovering therapeutic targets for HIV-1 transcription, targets for the post-transcriptional regulation of HIV-1 infections have received less attention. However, cumulative evidence now suggests the pivotal contribution of post-transcriptional regulation to the viral latency in both in vitro models and infected individuals. In this review, we explore recent insights on post-transcriptional latency in HIV-1 and discuss the potential of its therapeutic targets, illustrating some host factors that restrict HIV-1 at the post-transcriptional level.

Novel role of host protein SLC25A42 in the HIV-1 reactivation of latent HIV-1 provirus.

Despite the effectiveness of combination antiretroviral therapy, human immunodeficiency virus (HIV)infection remains incurable. To seek new strategies to overcome HIV type 1 (HIV-1) latency, one of the major barriers to HIV elimination, it is crucial to better understand how this state is maintained. Here, by means of an RNA interference screen employing an HIV-1 latency model using monocytic cell lines, we identified solute carrier family 25 member 42 (SLC25A42) as a potential host factor not previously known to affect HIV-1 latency. SLC25A42 knockdown resulted in increased HIV-1 expression, whereas forced expression of exogenous SLC25A42 suppressed it in SLC25A42-depleted cells. SLC25A42 depletion increased HIV-1 proviral transcriptional elongation but did not cause HIV-1 activation in an HIV-1 Tat-depleted latency model. This suggests that the role of SLC25A42 in HIV-1 transcription depends on HIV-1 Tat. Chromatin immunoprecipitation-qPCR analysis further revealed that SLC25A42 accumulated on or near the HIV-1 5' long terminal repeat promoter region of the HIV-1 provirus, suggesting a possible role in regulating HIV-1 Tat near this promoter region. These results indicate that SLC25A42 plays a novel role in HIV-1 latency maintenance in monocytic HIV-1 reservoirs.

Wogonin inhibits latent HIV-1 reactivation by downregulating histone crotonylation

BackgroundWogonin, a flavone isolated from Scutellaria baicalensis Georgi, is a commonly used phytochemical with anti-inflammatory and antitumor properties. However, the antiviral activity of wogonin against human immunodeficiency virus type 1 (HIV-1) has not been reported. PurposeThe current study aimed to explore whether wogonin can suppress latent HIV-1 reactivation and the mechanism of wogonin in inhibiting proviral HIV-1 transcription. MethodsWe assessed the effects of wogonin on HIV-1 reactivation using flow cytometry, cytotoxicity assay, quantitative PCR (qPCR), viral quality assurance (VQA), and western blot analysis. ResultsWogonin, a flavone isolated from S. baicalensis, significantly inhibited the reactivation of latent HIV-1 in cellular models and in primary CD4+ T cells from antiretroviral therapy (ART)-suppressed individuals ex vivo. Wogonin exhibited low cytotoxicity and long-lasting inhibition of HIV-1 transcription. Triptolide is a latency-promoting agent (LPA) that inhibits HIV-1 transcription and replication; wogonin had a stronger ability to inhibit HIV-1 latent reactivation than triptolide. Mechanistically, wogonin inhibited the reactivation of latent HIV-1 by inhibiting the expression of p300, a histone acetyltransferase, and decreasing the crotonylation of histone H3/H4 in the HIV-1 promoter region. ConclusionOur study found that wogonin is a novel LPA that can inhibit HIV-1 transcription by HIV-1 epigenetic silencing, which could bear promising significance for future applications of HIV-1 functional cure.

Novel perspectives on antisense transcription in HIV-1, HTLV-1, and HTLV-2.

The genome of retroviruses contains two promoter elements (called long terminal repeat or LTR) at the 5' and 3' end of their genome. Although the expression of retroviral genes generally depends on the promoter located in the 5' LTR, the 3' LTR also has promoter activity responsible for producing antisense transcripts. These natural antisense transcripts (NATs) are a class of RNA molecules transcribed from the opposite strand of a protein-coding gene. NATs have been identified in many prokaryotic and eukaryotic systems, as well as in human retroviruses such as human immunodeficiency virus type 1 (HIV-1) and HTLV-1/2 (human T-cell leukemia virus type 1/2). The antisense transcripts of HIV-1, HTLV-1, and HTLV-2 have been briefly characterized over the past several years. However, a complete appreciation of the role these transcripts play in the virus lifecycle and the cellular factors which regulate their transcription is still lacking. This review provides an overview of antisense transcription in human retroviruses with a specific focus on the MEF-2 family of transcription factors, the function(s) of the antisense protein products, and the application of antisense transcription models in therapeutics against HIV-1 and HTLV-1 in the context of co-infection.

The Myeloid-Specific Transcription Factor PU.1 Upregulates Mannose Receptor Expression but Represses Basal Activity of the HIV-LTR Promoter.

Human mannose receptor 1 (MRC1) is a cell surface receptor expressed in macrophages and other myeloid cells that inhibits human immunodeficiency virus type 1 (HIV-1) particle release by tethering virions to producer cell membranes. HIV-1 counteracts MRC1 expression by inhibiting mrc1 transcription. Here, we investigated the mechanism of MRC1 downregulation in HIV-1-infected macrophages. We identified the myeloid cell-specific transcription factor PU.1 as critical for regulating MRC1 expression. In the course of our study, we recognized a complex interplay between HIV-1 Tat and PU.1 transcription factors: Tat upregulated HIV-1 gene expression but inhibited mrc1 transcription, whereas PU.1 inhibited HIV-1 transcription but activated MRC1 expression. Disturbing this equilibrium by silencing PU.1 resulted in increased HIV-1 gene expression and reduced MRC1 promoter activity. Our study identified PU.1 as a central player in transcriptional control, regulating a complex interplay between viral and host gene expression in HIV-infected macrophages. IMPORTANCE HIV-1 replication in primary human cells depends on the activity of virus-encoded proteins but also involves cellular factors that can either promote (viral dependency factors) or inhibit (host restriction factors) virus replication. In previous work, we identified human MRC1 as a macrophage-specific host restriction factor that inhibits the detachment of viral particles from infected cells. Here, we report that HIV-1 counteracts this effect of MRC1 by imposing a transcriptional block on cellular MRC1 gene expression. The transcriptional inhibition of the MRC1 gene is accomplished by Tat, an HIV-1 factor whose best-described function actually is the enhancement of HIV-1 gene expression. Thus, HIV-1 has evolved to use the same protein for (i) activation of its own gene expression while (ii) inhibiting expression of MRC1 and other host factors.

Induction of Autophagy to Achieve a Human Immunodeficiency Virus Type 1 Cure.

Effective antiretroviral therapy has led to significant human immunodeficiency virus type 1 (HIV-1) suppression and improvement in immune function. However, the persistence of integrated proviral DNA in latently infected reservoir cells, which drive viral rebound post-interruption of antiretroviral therapy, remains the major roadblock to a cure. Therefore, the targeted elimination or permanent silencing of this latently infected reservoir is a major focus of HIV-1 research. The most studied approach in the development of a cure is the activation of HIV-1 expression to expose latently infected cells for immune clearance while inducing HIV-1 cytotoxicity—the “kick and kill” approach. However, the complex and highly heterogeneous nature of the latent reservoir, combined with the failure of clinical trials to reduce the reservoir size casts doubt on the feasibility of this approach. This concern that total elimination of HIV-1 from the body may not be possible has led to increased emphasis on a “functional cure” where the virus remains but is unable to reactivate which presents the challenge of permanently silencing transcription of HIV-1 for prolonged drug-free remission—a “block and lock” approach. In this review, we discuss the interaction of HIV-1 and autophagy, and the exploitation of autophagy to kill selectively HIV-1 latently infected cells as part of a cure strategy. The cure strategy proposed has the advantage of significantly decreasing the size of the HIV-1 reservoir that can contribute to a functional cure and when optimised has the potential to eradicate completely HIV-1.

The CREB Regulated Transcription Coactivator 2 Suppresses HIV-1 Transcription by Preventing RNA Pol II from Binding to HIV-1 LTR.

The CREB-regulated transcriptional co-activators (CRTCs), including CRTC1, CRTC2 and CRTC3, enhance transcription of CREB-targeted genes. In addition to regulating host gene expression in response to cAMP, CRTCs also increase the infection of several viruses. While human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter harbors a cAMP response element and activation of the cAMP pathway promotes HIV-1 transcription, it remains unknown whether CRTCs have any effect on HIV-1 transcription and HIV-1 infection. Here, we reported that CRTC2 expression was induced by HIV-1 infection, but CRTC2 suppressed HIV-1 infection and diminished viral RNA expression. Mechanistic studies revealed that CRTC2 inhibited transcription from HIV-1 LTR and diminished RNA Pol II occupancy at the LTR independent of its association with CREB. Importantly, CRTC2 inhibits the activation of latent HIV-1.Together, these data suggest that in response to HIV-1 infection, cells increase the expression of CRTC2 which inhibits HIV-1 gene expression and may play a role in driving HIV-1 into latency.

Balance between Retroviral Latency and Transcription: Based on HIV Model.

The representative of the Lentivirus genus is the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). To date, there is no cure for AIDS because of the existence of the HIV-1 reservoir. HIV-1 infection can persist for decades despite effective antiretroviral therapy (ART), due to the persistence of infectious latent viruses in long-lived resting memory CD4+ T cells, macrophages, monocytes, microglial cells, and other cell types. However, the biology of HIV-1 latency remains incompletely understood. Retroviral long terminal repeat region (LTR) plays an indispensable role in controlling viral gene expression. Regulation of the transcription initiation plays a crucial role in establishing and maintaining a retrovirus latency. Whether and how retroviruses establish latency and reactivate remains unclear. In this article, we describe what is known about the regulation of LTR-driven transcription in HIV-1, that is, the cis-elements present in the LTR, the role of LTR transcription factor binding sites in LTR-driven transcription, the role of HIV-1-encoded transactivator protein, hormonal effects on virus transcription, impact of LTR variability on transcription, and epigenetic control of retrovirus LTR. Finally, we focus on a novel clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/dCas9)-based strategy for HIV-1 reservoir purging.

Broadly active zinc finger protein-guided transcriptional activation of HIV-1

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the “shock-and-kill” approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.

The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription.

The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.

Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

Modeling of the HIV-1 Life Cycle in Productively Infected Cells to Predict Novel Therapeutic Targets.

There are many studies that model the within-host population dynamics of Human Immunodeficiency Virus Type 1 (HIV-1) infection. However, the within-infected-cell replication of HIV-1 remains to be not comprehensively addressed. There exist rather few quantitative models describing the regulation of the HIV-1 life cycle at the intracellular level. In treatment of HIV-1 infection, there remain issues related to side-effects and drug-resistance that require further search “...for new and better drugs, ideally targeting multiple independent steps in the HIV-1 replication cycle” (as highlighted recently by Tedbury & Freed, The Future of HIV-1 Therapeutics, 2015). High-resolution mathematical models of HIV-1 growth in infected cells provide an additional analytical tool in identifying novel drug targets. We formulate a high-dimensional model describing the biochemical reactions underlying the replication of HIV-1 in target cells. The model considers a nonlinear regulation of the transcription of HIV-1 mediated by Tat and the Rev-dependent transport of fully spliced and singly spliced transcripts from the nucleus to the cytoplasm. The model is calibrated using available information on the kinetics of various stages of HIV-1 replication. The sensitivity analysis of the model is performed to rank the biochemical processes of HIV-1 replication with respect to their impact on the net production of virions by one actively infected cell. The ranking of the sensitivity factors provides a quantitative basis for identifying novel targets for antiviral therapy. Our analysis suggests that HIV-1 assembly depending on Gag and Tat-Rev regulation of transcription and mRNA distribution present two most critical stages in HIV-1 replication that can be targeted to effectively control virus production. These processes are not covered by current antiretroviral treatments.

Human immunodeficiency virus type 1 transcription is regulated by thieno[3,4- d ]pyrimidine.

In the present study, the effect of thieno[3,4-d]pyrimidine (TEP) on the transcription of human immunodeficiency virus type 1 (HIV-1) was investigated. To the best of the authors' knowledge, this is the first study describing the effect of TEP on the transcription of HIV-1. The present results identified a marked decrease in the production of the HIV-1 genome in 293T cells after treatment with TEP. The treatment of HIV-1infected 293T cells with TEP led to the upregulation of retinoblastoma binding protein 4 (RbAp48) mRNA and protein. The activity of long terminal repeats (LTRs) was decreased by 19, 24, 29, 34, 38, 41, 52, 63, 76 and 92% in treatments with concentrations of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25 and 2.5 µM TEP, respectively. The p65 translocation to the nucleus was markedly reduced in 293T cells treated with TEP for 48 h. A marked decrease was observed in the production of HIV-1 in 293T cells with the increase in concentration of pRbAp48. In 293T cells, RbAp48 and TEP decreased tumor necrosis factor-α and phorbol 12-myristate 13-acetate-induced activity of LTR. Therefore, the present study suggested that TEP inhibited transcription of HIV-1 through upregulation of RbAp48 expression and activation of the NF-κB pathway. Therefore, TEP may be used for the treatment of HIV-1 infection.

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.

Chromatin maturation of the HIV-1 provirus in primary resting CD4+T cells.

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.

Identification of novel compounds against Tat-mediated human immunodeficiency virus-1 transcription by high-throughput functional screening assay

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2′-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase.Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

Fimepinostat, a novel dual inhibitor of HDAC and PI3K, effectively reverses HIV-1 latency ex vivo without T cell activation

ObjectivesTo test the potential of fimepinostat (CUDC-907), a dual inhibitor of histone deacetylases (HDAC) and phosphatidylinositol-3-kinases (PI3K), to reverse human immunodeficiency virus type 1 (HIV-1) latency in infected cell lines and in CD4+ T cells from HIV-1-infected donors on long-term combination antiretroviral therapy (cART). MethodsLatently HIV-1-infected J-lat Tat-GFP and ACH-2 cell lines were stimulated with clinically relevant concentrations of fimepinostat using the HDAC inhibitors (HDACi) panobinostat and romidepsin for comparison. Next, CD4+ T cells from donors living with HIV-1 on long-term cART were stimulated ex vivo and cell-associated unspliced HIV-1 RNA was measured to quantify changes in HIV-1 transcription. Finally, the impact of fimepinostat on T cell activation (CD69 expression) and proliferation (Ki67 expression) was determined using peripheral blood mononuclear cells from uninfected donors. ResultsWe found fimepinostat to be a potent latency-reversing agent. This was true in two latently infected cell lines as well as ex vivo in CD4+ T cells isolated from donors living with HIV-1. Relative to therapeutic dosing levels, fimepinostat showed latency-reversing potential comparable to romidepsin, which is the most potent HDACi tested in HIV-1 cure-related trials. Interestingly, in contrast to romidepsin, fimepinostat stimulation resulted in decreased T cell activation and had no negative impact on T cell proliferation. ConclusionsAt therapeutic concentration, the dual HDAC and PI3K inhibitor fimepinostat was a potent HIV-1 latency-reversing agent and it did not induce T cell activation and proliferation. The potential of fimepinostat as a latency-reversing agent warrants further investigation.

Interferon-inducible TRIM22 contributes to maintenance of HIV-1 proviral latency in T cell lines

The human immunodeficiency virus type-1 (HIV-1) establishes a state of latent infection in a small number of CD4+ T lymphocytes that, nonetheless, represent a major obstacle to viral eradication. We here show that Tripartite Motif-containing protein 22 (TRIM22), an epigenetic inhibitor of Specificity protein 1 (Sp1)-dependent HIV-1 transcription, is a relevant factor in maintaining a state of repressed HIV-1 expression at least in CD4+ T cell lines. By knocking-down (KD) TRIM22 expression, we observed an accelerated reactivation of a doxycycline (Dox)-controlled HIV-1 replication in the T lymphocytic SupT1 cell line. Furthermore, we here report for the first time that TRIM22 is a crucial factor for maintaining a state of HIV-1 quiescence in chronically infected ACH2 -T cell line while its KD potentiated HIV-1 expression in both ACH-2 and J-Lat 10.6 cell lines upon cell stimulation with either tumor necrosis factor-α (TNF-α) or histone deacetylase inhibitors (HDACi). In conclusion, TRIM22 is a novel determinant of HIV-1 latency, at least in T cell lines, thus representing a potential pharmacological target for strategies aiming at curtailing or silencing the pool of latently infected CD4+ T lymphocytes constituting the HIV-1 reservoir in individuals receiving combination antiretroviral therapy.

USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1.

The host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase. Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+ T cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by downregulating p21 expression and upregulating intracellular dNTP levels. p21 downregulation by USP18 was associated with the active form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.IMPORTANCE Macrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1, mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The cyclin-dependent kinase inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 downregulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

Curaxin CBL0100 Blocks HIV-1 Replication and Reactivation through Inhibition of Viral Transcriptional Elongation.

Despite combination antiretroviral therapy (cART), acquired immunodeficiency syndrome (AIDS), predominantly caused by the human immunodeficiency virus type 1 (HIV-1), remains incurable. The barrier to a cure lies in the virus' ability to establish a latent infection in HIV/AIDS patients. Unsurprisingly, efforts for a sterilizing cure have focused on the “shock and kill” strategy using latency-reversing agents (LRAs) to complement cART in order to eliminate these latent reservoirs. However, this method faces numerous challenges. Recently, the “block and lock” strategy has been proposed. It aims to reinforce a deep state of latency and prevent sporadic reactivation (“blip”) of HIV-1 using latency-promoting agents (LPAs) for a functional cure. Our studies of curaxin 100 (CBL0100), a small-molecule targeting the facilitates chromatin transcription (FACT) complex, show that it blocks both HIV-1 replication and reactivation in in vitro and ex vivo models of HIV-1. Mechanistic investigation elucidated that CBL0100 preferentially targets HIV-1 transcriptional elongation and decreases the occupancy of RNA Polymerase II (Pol II) and FACT at the HIV-1 promoter region. In conclusion, CBL0100 is a newly identified inhibitor of HIV-1 transcription that can be used as an LPA in the “block and lock” cure strategy.