HIV-1 Promoter Research Articles

HIV-1 transcriptional silencing caused by TRIM22 inhibition of Sp1 binding to the viral promoter.

BackgroundIntracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR.Findings293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4+ SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays.ConclusionTRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter.

Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells.

The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency.

ABSTRACTAntiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4+ T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4+ T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs.

The Werner Protein Acts as a Coactivator of Nuclear Factor κB (NF-κB) on HIV-1 and Interleukin-8 (IL-8) Promoters

The Werner syndrome helicase (WRN) plays a role in maintaining genomic stability. The lack of WRN results in Werner syndrome, a rare autosomal recessive genetic disorder, which causes premature aging accompanied by many complications such as rare forms of cancer and type 2 diabetes. However, the underlying mechanisms of these complications, arising due to the loss of WRN, are poorly understood. In this study, we demonstrated the function of WRN in transcriptional regulation of NF-κB targets. WRN physically interacts via its RecQ C-terminal (RQC) domain with the Rel homology domain of both the RelA (p65) and the p50 subunits of NF-κB. In the steady state, WRN is recruited to HIV-1 long terminal repeat (LTR), a typical NF-κB-responsive promoter, as well as the p50/p50 homodimer, in an NF-κB site-dependent manner. The amount of WRN on LTR increased along with the transactivating RelA/p50 heterodimer in response to TNF-α stimulation. Further, a knockdown of WRN reduced the transactivation of LTR in exogenous RelA/p50-introduced or TNF-α-stimulated cells. Additionally, knockdown of WRN reduced TNF-α stimulation-induced activation of the endogenous promoter of IL-8, an NF-κB-responsive gene, and WRN increased its association with the IL-8 promoter region together with RelA/p50 after TNF-α stimulation. In conjunction with studies that have shown NF-κB to be a key regulator of aging and inflammation, our results indicate a novel role of WRN in transcriptional regulation. Along with NF-κB, the loss of WRN is expected to result in incorrect regulation of downstream targets and leads to immune abnormalities and homeostatic disruption.

H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4 + T Cells

Histone methyltransferase inhibitors (HMTis) and histone deacetylase inhibitors (HDACis) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV-1), but studies have largely been performed with cell lines. As specific and potent HMTis directed at EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 1)/EZH2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 inhibitor, reduced trimethylation of histone 3 at lysine 27 (H3K27) of the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells. However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in increases in HIV gag RNA and HIV p24 antigen production that were up to 2.5-fold greater than those induced by VOR alone. Therefore, in primary resting CD4(+) T cells, true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at nucleosome 1 (Nuc-1). Based upon our findings, the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials. Demethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow, occurring over 96 h, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4(+) T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection.

The anti-inflammatory activity of curcumin protects the genital mucosal epithelial barrier from disruption and blocks replication of HIV-1 and HSV-2.

Inflammation is a known mechanism that facilitates HIV acquisition and the spread of infection. In this study, we evaluated whether curcumin, a potent and safe anti-inflammatory compound, could be used to abrogate inflammatory processes that facilitate HIV-1 acquisition in the female genital tract (FGT) and contribute to HIV amplification. Primary, human genital epithelial cells (GECs) were pretreated with curcumin and exposed to HIV-1 or HIV glycoprotein 120 (gp120), both of which have been shown to disrupt epithelial tight junction proteins, including ZO-1 and occludin. Pre-treatment with curcumin prevented disruption of the mucosal barrier by maintaining ZO-1 and occludin expression and maintained trans-epithelial electric resistance across the genital epithelium. Curcumin pre-treatment also abrogated the gp120-mediated upregulation of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6, which mediate barrier disruption, as well as the chemokines IL-8, RANTES and interferon gamma-induced protein-10 (IP-10), which are capable of recruiting HIV target cells to the FGT. GECs treated with curcumin and exposed to the sexually transmitted co-infecting microbes HSV-1, HSV-2 and Neisseria gonorrhoeae were unable to elicit innate inflammatory responses that indirectly induced activation of the HIV promoter and curcumin blocked Toll-like receptor (TLR)-mediated induction of HIV replication in chronically infected T-cells. Finally, curcumin treatment resulted in significantly decreased HIV-1 and HSV-2 replication in chronically infected T-cells and primary GECs, respectively. All together, our results suggest that the use of anti-inflammatory compounds such as curcumin may offer a viable alternative for the prevention and/or control of HIV replication in the FGT.

SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication.

Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by >200-fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA-binding domains but not the arginine serine-rich activator domain can downregulate viral replication by >2,000-fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA-binding domains. Most drugs utilized to treat the HIV-1 infection are based on compounds that directly target proteins encoded by the virus. However, given the high viral mutation rate, the appearance of novel drug-resistant viral strains is common. Thus, there is a need for novel therapeutics with diverse mechanisms of action. In this study, we show that the cellular protein SRSF1 is a strong inhibitor of viral replication. Furthermore, expression of the SRSF1 RNA-binding domains alone can inhibit viral replication by >2,000-fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein, the RNA-binding domains, and the minimal effects observed on cell metabolism, further studies are warranted to assess the therapeutic potential of peptides derived from these sequences.

Role of mannose-binding lectin deficiency in HIV-1 and schistosoma infections in a rural adult population in Zimbabwe.

BackgroundPolymorphism in the MBL2 gene lead to MBL deficiency, which has been shown to increase susceptibility to various bacterial, viral and parasitic infections. We assessed role of MBL deficiency in HIV-1 and schistosoma infections in Zimbabwean adults enrolled in the Mupfure Schistosomiasis and HIV Cohort (MUSH Cohort).MethodsHIV-1, S. haematobium and S. mansoni infections were determined at baseline. Plasma MBL concentration was measured by ELISA and MBL2 genotypes determined by PCR. We calculated and compared the proportions of plasma MBL deficiency, MBL2 structural variant alleles B (codon 54A>G), C (codon 57A>G), and D (codon 52T>C) as well as MBL2 promoter variants -550(H/L), -221(X/Y) and +4(P/Q) between HIV-1 and schistosoma co-infection and control groups using Chi Square test.ResultsWe assessed 379 adults, 80% females, median age (IQR) 30 (17–41) years. HIV-1, S. haematobium and S. mansoni prevalence were 26%, 43% and 18% respectively in the MUSH baseline survey. Median (IQR) plasma MBL concentration was 800μg/L (192-1936μg/L). Prevalence of plasma MBL deficiency was 18% with high frequency of the C (codon 57G>A) mutant allele (20%). There was no significant difference in median plasma MBL levels between HIV negative (912μg/L) and HIV positive (688μg/L), p = 0.066. However plasma MBL levels at the assay detection limit of 20μg/L were more frequent among the HIV-1 infected (p = 0.007). S. haematobium and S. mansoni infected participants had significantly higher MBL levels than uninfected. All MBL2 variants were not associated with HIV-1 infection but promoter variants LY and LL were significantly associated with S. haematobium infection.ConclusionOur data indicate high prevalence of MBL deficiency, no evidence of association between MBL deficiency and HIV-1 infection. However, lower plasma MBL levels were protective against both S. haematobium and S. mansoni infections and MBL2 promoter and variants LY and LL increased susceptibility to S. haematobium infection.

Molecular mechanisms linking high dose medroxyprogesterone with HIV-1 risk.

BackgroundEpidemiological studies suggest that medroxyprogesterone acetate (MPA) may increase the risk of HIV-1. The current studies were designed to identify potential underlying biological mechanisms.MethodsHuman vaginal epithelial (VK2/E6E7), peripheral blood mononuclear (PBMC), and polarized endometrial (HEC-1-A) cells were treated with a range of concentrations of MPA (0.015-150 μg/ml) and the impact on gene expression, protein secretion, and HIV infection was evaluated.ResultsTreatment of VK2/E6E7 cells with high doses (>15μg/ml] of MPA significantly upregulated proinflammatory cytokines, which resulted in a significant increase in HIV p24 levels secreted by latently infected U1 cells following exposure to culture supernatants harvested from MPA compared to mock-treated cells. MPA also increased syndecan expression by VK2/E6E7 cells and cells treated with 15 μg/ml of MPA bound and transferred more HIV-1 to T cells compared to mock-treated cells. Moreover, MPA treatment of epithelial cells and PBMC significantly decreased cell proliferation resulting in disruption of the epithelial barrier and decreased cytokine responses to phytohaemagglutinin, respectively.ConclusionWe identified several molecular mechanisms that could contribute to an association between DMPA and HIV including proinflammatory cytokine and chemokine responses that could activate the HIV promoter and recruit immune targets, increased expression of syndecans to facilitate the transfer of virus from epithelial to immune cells and decreased cell proliferation. The latter could impede the ability to maintain an effective epithelial barrier and adversely impact immune cell function. However, these responses were observed primarily following exposure to high (15-150 μg/ml) MPA concentrations. Clinical correlation is needed to determine whether the prolonged MPA exposure associated with contraception activates these mechanisms in vivo.

BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection.

BackgroundViruses have naturally evolved elegant strategies to manipulate the host’s cellular machinery, including ways to hijack cellular DNA repair proteins to aid in their own replication. Retroviruses induce DNA damage through integration of their genome into host DNA. DNA damage signaling proteins including ATR, ATM and BRCA1 contribute to multiple steps in the HIV-1 life cycle, including integration and Vpr-induced G2/M arrest. However, there have been no studies to date regarding the role of BRCA1 in HIV-1 transcription.MethodsHere we performed various transcriptional analyses to assess the role of BRCA1 in HIV-1 transcription by overexpression, selective depletion, and treatment with small molecule inhibitors. We examined association of Tat and BRCA1 through in vitro binding assays, as well as BRCA1-LTR association by chromatin immunoprecipitation.ResultsBRCA1 was found to be important for viral transcription as cells that lack BRCA1 displayed severely reduced HIV-1 Tat-dependent transcription, and gain or loss-of-function studies resulted in enhanced or decreased transcription. Moreover, Tat was detected in complex with BRCA1 aa504-802. Small molecule inhibition of BRCA1 phosphorylation effector kinases, ATR and ATM, decreased Tat-dependent transcription, whereas a Chk2 inhibitor showed no effect. Furthermore, BRCA1 was found at the viral promoter and treatment with curcumin and ATM inhibitors decreased BRCA1 LTR occupancy. Importantly, these findings were validated in a highly relevant model of HIV infection and are indicative of BRCA1 phosphorylation affecting Tat-dependent transcription.ConclusionsBRCA1 presence at the HIV-1 promoter highlights a novel function of the multifaceted protein in HIV-1 infection. The BRCA1 pathway or enzymes that phosphorylate BRCA1 could potentially be used as complementary host-based treatment for combined antiretroviral therapy, as there are multiple potent ATM inhibitors in development as chemotherapeutics.

Delivery of behavioral HIV prevention services in New York City outpatient substance abuse treatment clinics: providers' perspectives on opportunities and challenges.

Providers (e.g., counselors, physicians) of substance abuse treatment have an opportunity to address HIV. This study identified: (1) providers' HIV prevention practices, (2) barriers, and (3) promoters to offering HIV prevention in substance abuse treatment. Semistructured qualitative interviews with one director, one medical provider, and four counselors, from each of six outpatient clinics (N = 36) were transcribed and coded according to thematic content analysis. Providers' practices included: (1) recommending condoms, (2) explaining HIV transmission, (3) HIV testing, and (4) assessing risk. Barriers included: (1) believing that clients know enough about HIV, (2) believing that clients are not at risk, (3) lacking information, (4) outdated training (i.e., > 5 years ago), (5) HIV stigma, and (6) avoidance. While some providers recommended condoms and HIV testing, many avoided discussing HIV. Our results suggest a need for training to improve understanding of HIV transmission, effective counseling practices, and to build capacity for HIV testing or linkages with HIV service agencies.

Promoter Targeting RNAs: Unexpected Contributors to the Control of HIV-1 Transcription

In spite of prolonged and intensive treatment with combined antiretroviral therapy (cART), which efficiently suppresses plasma viremia, the integrated provirus of HIV-1 persists in resting memory CD4+ T cells as latent infection. Treatment with cART does not substantially reduce the burden of latent infection. Once cART is ceased, HIV-1 replication recrudesces from these reservoirs in the overwhelming majority of patients. There is increasing evidence supporting a role for noncoding RNAs (ncRNA), including microRNAs (miRNAs), antisense (as)RNAs, and short interfering (si)RNA in the regulation of HIV-1 transcription. This appears to be mediated by interaction with the HIV-1 promoter region. Viral miRNAs have the potential to act as positive or negative regulators of HIV transcription. Moreover, inhibition of virally encoded long-asRNA can induce positive transcriptional regulation, while antisense strands of siRNA targeting the NF-κB region suppress viral transcription. An in-depth understanding of the interaction between ncRNAs and the HIV-1 U3 promoter region may lead to new approaches for the control of HIV reservoirs. This review focuses on promoter associated ncRNAs, with particular emphasis on their role in determining whether HIV-1 establishes active or latent infection.

Designed transcription activator-like effector proteins efficiently induced the expression of latent HIV-1 in latently infected cells.

HIV latency is the foremost barrier to clearing HIV infection from patients. Reactivation of latent HIV-1 represents a promising strategy to deplete these viral reservoirs. Here, we report a novel approach to reactivate latent HIV-1 provirus using artificially designed transcription activator-like effector (TALE) fusion proteins containing a DNA-binding domain specifically targeting the HIV-1 promoter and the herpes simplex virus-based transcriptional activator VP64 domain. We engineered four TALE genes (TALE1-4) encoding TALE proteins, each specifically targeting different 20-bp DNA sequences within the HIV-1 promoter, and we constructed four TALE-VP64 expression vectors corresponding to TALE1-4. We found that TALE1-VP64 effectively reactivated HIV-1 gene expression in latently infected C11 and A10.6 cells. We further confirmed that TALE1-VP64 reactivated latent HIV-1 via specific binding to the HIV-LTR promoter. Moreover, we also found that TALE1-VP64 did not affect cell proliferation or cell cycle distribution. Taken together, our data demonstrated that TALE1-VP64 can specifically and effectively reactivate latent HIV-1 transcription, suggesting that this strategy may provide a novel approach for anti-HIV-1 latency therapy in the future.

The integrase cofactor LEDGF/p75 associates with Iws1 and Spt6 for postintegration silencing of HIV-1 gene expression in latently infected cells.

The persistence of a latent reservoir containing transcriptionally silent, but replication-competent, integrated provirus is a serious challenge to HIV eradication. HIV integration is under the control of LEDGF/p75, the cellular cofactor of viral integrase. Investigating possible postintegration roles for LEDGF/p75, we find that LEDGF/p75 represses HIV expression in latently infected cells. LEDGF/p75 associated with two proteins involved in the control of gene expression and chromatin structure, Spt6 and Iws1, to form a stable complex. Iws1 plays a role in the establishment of latent infection, whereas Spt6 functions to recruit Iws1 and LEDGF/p75 to the silenced provirus and maintains histone occupancy at the HIV promoter. In latently infected cells, depletion of the complex results in reactivation of HIV expression Altogether, our results indicate that a complex containing LEDGF/p75, Iws1, and Spt6 participates in regulating postintegration steps of HIV latency.

Cladograms with Path to Event (ClaPTE): A novel algorithm to detect associations between genotypes or phenotypes using phylogenies

BackgroundAssociations between genotype and phenotype provide insight into the evolution of pathogenesis, drug resistance, and the spread of pathogens between hosts. However, common ancestry can lead to apparent associations between biologically unrelated features. The novel method Cladograms with Path to Event (ClaPTE) detects associations between character-pairs (either a pair of mutations or a mutation paired with a phenotype) while adjusting for common ancestry, using phylogenetic trees. MethodsClaPTE tests for character-pairs changing close together on the phylogenetic tree, consistent with an associated character-pair. ClaPTE is compared to three existing methods (independent contrasts, mixed model, and likelihood ratio) to detect character-pair associations adjusted for common ancestry. Comparisons utilize simulations on gene trees for: HIV Env, HIV promoter, and bacterial DnaJ and GuaB; and case studies for Oseltamavir resistance in Influenza, and for DnaJ and GuaB. Simulated data include both true-positive/associated character-pairs, and true-negative/not-associated character-pairs, used to assess type I (frequency of p-values in true-negatives) and type II (sensitivity to true-positives) error control. Results and conclusionsClaPTE has competitive sensitivity and better type I error control than existing methods. In the Influenza/Oseltamavir case study, ClaPTE reports no new permissive mutations but detects associations between adjacent (in primary sequence) amino acid positions which other methods miss. In the DnaJ and GuaB case study, ClaPTE reports more frequent associations between positions both from the same protein family than between positions from different families, in contrast to other methods. In both case studies, the results from ClaPTE are biologically plausible.

The HOPE social media intervention for global HIV prevention in Peru: a cluster randomised controlled trial

Social media technologies offer new approaches to HIV prevention and promotion of testing. We examined the efficacy of the Harnessing Online Peer Education (HOPE) social media intervention to increase HIV testing among men who have sex with men (MSM) in Peru. In this cluster randomised controlled trial, Peruvian MSM from Greater Lima (including Callao) who had sex with a man in the past 12 months, were 18 years of age or older, were HIV negative or serostatus unknown, and had a Facebook account or were willing to create one (N=556) were randomly assigned (1:1) by concealed allocation to join intervention or control groups on Facebook for 12 weeks. For the intervention, Peruvian MSM were trained and assigned to be HIV prevention mentors (peer-leaders) to participants in Facebook groups. The interventions period lasted 12 weeks. Participants in control groups received an enhanced standard of care, including standard offline HIV prevention available in Peru and participation in Facebook groups (without peer leaders) that provided study updates and HIV testing information. After accepting a request to join the groups, continued participation was voluntary. Participants also completed questionnaires on HIV risk behaviours and social media use at baseline and 12 week follow-up. The primary outcome was the number of participants who received a free HIV test at a local community clinic. The facebook groups were analysed as clusters to account for intracluster correlations. This trial is registered with ClinicalTrials.gov, number NCT01701206. Of 49 peer-leaders recruited, 34 completed training and were assigned at random to the intervention Facebook groups. Between March 19, 2012, and June 11, 2012, and Sept 26, 2012, and Dec 19, 2012, 556 participants were randomly assigned to intervention groups (N=278) or control groups (N=278); we analyse data for 252 and 246. 43 participants (17%) in the intervention group and 16 (7%) in the control groups got tested for HIV (adjusted odds ratio 2·61, 95% CI 1·55–4·38). No adverse events were reported. Development of peer-mentored social media communities seemed to be an efficacious method to increase HIV testing among high-risk populations in Peru. Results suggest that the HOPE social media intervention could improve HIV testing rates among MSM in Peru. National Institute of Mental Health.

Epigenetic regulation of inflammatory gene expression in macrophages by selenium

Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trspfl/flCreLysM mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.

HIV Excision Utilizing CRISPR/Cas9 Technology: Attacking the Proviral Quasispecies in Reservoirs to Achieve a Cure.

Recently several gene-editing technologies developed are being explored for their potential utility in providing new and unique treatments for HIV. One of these technologies is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 system. This system is being explored for its utility against host genes important to HIV infection, namely the HIV coreceptor CCR5, and for excision of the integrated genome from infected cells by targeting selected genes or genomic regions, especially the HIV-1 promoter or long terminal repeat (LTR). One of the major hurdles with the development of this technology for use in patients is defining the LTR sequence spectrum within the viral quasispecies present in the integrated virus and how that effects the number of guide RNAs (gRNAs) required to completely excise all proviral genomes. In this study, the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort was utilized to demonstrate that [1] the predominant sequence of the integrated proviral LTR within the PBMC compartment shows a decrease in the amount of variation per year regardless of the type of therapy; [2] predominant HIV-1 LTR sequence undergoes continued genetic change with respect to the predominant genotype in these cells for at least 6 years while on effective suppressive ART; [3] using next generation sequencing (NGS), to demonstrate that 4 of the 8 patient samples examined could have a complete gRNA regimen designed to target all known quasispecies; and [4] length of HAART therapy may reduce the number of gRNA required to eradicate provirus as shown by NGS and gRNA design for longitudinal samples of patient A0017 in the CARES cohort. Overall, these studies demonstrate the feasibility of addressing at least one of the major technological challenges of CRISPR/Cas9-mediated HIV-1 proviral genome eradication involving the effective targeting of all viral quasispecies in a given patient sample.

A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation.

HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P-RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P-S2P transition at the integrated HIV-1 promoter.