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- Role of cellular iron and oxygen in the regulation of HIV-1 infection. [JOURNAL ARTICLE]
- Future Virol 2013 Mar; 8(3):301-311.
Despite efficient antiretroviral therapy, eradication of HIV-1 infection is challenging and requires novel biological insights and therapeutic strategies. Among other physiological and environmental factors, intracellular iron greatly affects HIV-1 replication. Higher iron stores were shown to be associated with faster progression of HIV-1 infection and to inversely correlate with the survival of HIV-1 infected patients. Iron is required for several steps in the HIV-1 life cycle, including reverse transcription, HIV-1 gene expression and capsid assembly. Here, the authors present a comprehensive review of the molecular mechanisms involved in iron- and oxygen-mediated regulation of HIV-1 replication. We also propose key intracellular pathways that may be involved in regulating HIV-1 replication, via protein kinase complexes, CDK9/cyclin T1 and CDK 2/cyclin E, protein phosphatase-1 and other host factors.
- Viral suppression after 12 months of antiretroviral therapy in low- and middle-income countries: a systematic review. [Journal Article]
- Bull World Health Organ 2013 May 1; 91(5):377-385E.
To establish estimates of viral suppression in low- and middle-income countries (LMICs) in patients who received antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection.Data on viral suppression after 12 months of ART in LMICs were collected from articles published in 2003 to 2011 and from abstracts of conferences held between 2009 and 2011. Pooled proportions for on-treatment and intention-to-treat populations were used as summary estimates. Random-effects models were used for heterogeneous groups of studies (I (2) > 75%).Overall, 49 studies covering 48 cohorts and 30 016 individuals met the inclusion criteria. With thresholds for suppression between 300 and 500 copies of viral ribonucleic acid (RNA) per ml of plasma, 84.3% (95% confidence interval, CI: 80.4-87.9) of the pooled on-treatment population and 70.5% (95% CI: 65.2-75.6) of the intention-to-treat population showed suppression. Use of different viral RNA thresholds changed the proportions showing suppression: to 84% and 76% of the on-treatment population with thresholds set above 300 and at or below 200 RNA copies per ml, respectively, and to 78%, 71% and 63% of the intention-to-treat population at thresholds set at 1000, 300 to 500, and 200 or fewer copies per ml, respectively.The pooled estimates of viral suppression recorded after 12 months of ART in LMICs provide benchmarks that other ART programmes can use to set realistic goals and perform predictive modelling. Evidence from this review suggests that the current international target - i.e. viral suppression in > 70% of the intention-to-treat population, with a threshold of 1000 copies per ml - should be revised upwards.
- Productive entry of HIV-1 during cell-to-cell transmission via dynamin-dependent endocytosis. [JOURNAL ARTICLE]
- J Virol 2013 May 15.
HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4(+) T-cells is the more efficient mode of transmission and is predominant in lymphoid tissue where the majority of virus resides. Yet, the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can uptake virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be non-productive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and was sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4(+) T-cell lines and in primary CD4(+) T-cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways, as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner, but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin-accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters.
- Adaptor Protein-1 (AP-1) Promotes Cross-presentation Through the Same Tyrosine Signal in MHC-I that is Targeted by HIV-1. [JOURNAL ARTICLE]
- J Virol 2013 May 15.
Certain antigen presenting cells (APCs) process and present extracellular antigen with MHC class I molecules to activate naïve CD8(+) T cells in a process termed cross-presentation. We used insights gained from HIV immune evasion strategies to demonstrate that the clathrin adaptor protein, AP-1, is necessary for cross-presentation by MHC-I molecules containing a cytoplasmic tail tyrosine signal (murine MHC-I molecules, human MHC-I HLA-A and HLA-B allotypes). In contrast, AP-1 activity was not needed for cross-presentation by MHC-I molecules containing a human MHC-I HLA-C cytoplasmic tail, which does not contain a tyrosine signal. AP-1 activity was also dispensable for presentation of endogenous antigens by MHC-I via the classical pathway. In APCs, we show that HIV Nef disrupts cross-presentation by MHC-I containing the tyrosine signal but does not affect cross-presentation by MHC-I containing the HLA-C cytoplasmic tail. Thus, we provide evidence for two separable cross-presentation pathways, only one of which is targeted by HIV.
- Novel Inhibitors of SARS-CoV Entry acting by Three Distinct Mechanisms. [JOURNAL ARTICLE]
- J Virol 2013 May 15.
Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus, (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of small molecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S), but not with Vesicular Stomatitis Virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that they act by three distinct mechanisms: a) SSAA09E2 (N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide) acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, Angiotensin Converting Enzyme-2 (ACE2); b) SSAA09E1 ([(Z)-1-thiophen-2-ylethylideneamino]thiourea), which acts later by blocking cathepsin L, a host protease required for processing of SARS-S during viral entry and c) SSAA09E3 (N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide)), which also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L, but prevents fusion of the viral membrane with the host cellular membrane. Our work demonstrates that there are at least three independent strategies to block SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.
- HIV Populations are Large and Accumulate High Genetic Diversity in Nonlinear Fashion. [JOURNAL ARTICLE]
- J Virol 2013 May 15.
HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence, and to estimate replicating population sizes in a group of treatment naive HIV-infected individuals sampled at single (N=22) or multiple, longitudinal time points (N=11). Analysis of single genome sequences (SGS) revealed non-linear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were slow relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.
- Evaluation of Xpert MTB/RIF for the detection of TB from blood of HIV-infected adults confirms MTB-bacteremia as indicator of poor prognosis. [JOURNAL ARTICLE]
- J Clin Microbiol 2013 May 15.
Tuberculosis remains a leading cause of death in HIV infected adults, in part due to delay in diagnosis and therefore initiation of treatment. Recently the Gene-Xpert platform, a rapid, PCR based diagnostic platform has been validated for the diagnosis of TB using sputum. We have evaluated the Xpert MTB/RIF assay for diagnosis of MTB-bacteremia and investigated its impact on clinical outcomes. Consecutive HIV-infected adults with fever and cough presenting to Queen Elizabeth Central Hospital, Blantyre, Malawi were recruited and followed up for 2 months. At presentation, 3 sputa were examined by smear, culture and Xpert MTB/RIF assay for the presence of Mycobacterium tuberculosis and blood was drawn for PCR using Xpert, mycobacterial culture (Myco/F Lytic), aerobic culture. 104 patients were recruited and 44 (43%) were sputum culture positive for Mycobacterium tuberculosis. 10 were Xpert-blood positive, a sensitivity of 21% and specificity of 100%. Two week mortality was significantly higher amongst patients who were Xpert-blood positive when compared to those who were negative (40% vs. 3%, multivariate Odds Ratio for death if positive: 22 (95%CI: 3-174). This effect persisted on assessment of mortality at 2 months (40% vs. 11%, OR: 5.6, 95% CI: 1.3-24.6). When screening uncomplicated patients presenting with productive cough for pulmonary tuberculosis, Xpert-blood offers no diagnostic advantage over sputum. Despite this, Xpert-blood positivity is highly predictive of early mortality and this test rapidly identifies a group of patients in urgent need of initiation of treatment.
- mTOR Plays a Critical Role in p53-induced Oxidative Kidney Cell Injury in HIVAN. [JOURNAL ARTICLE]
- Am J Physiol Renal Physiol 2013 May 15.
Oxidative stress has been implicated to contribute to HIV-induced kidney cell injury; however, the role of p53, a modulator of oxidative stress, has not been evaluated in the development of HIV-associated nephropathy (HIVAN). We hypothesized that mammalian target of rapamycin (mTOR) may be critical for the induction of p53 mediated oxidative kidney cell injury in HIVAN. To test our hypothesis, we evaluated the effect of an mTOR inhibitor, rapamycin, on kidney cell p53 expression, down stream signaling, and kidney cell injury both in in vivo and in vitro studies. Inhibition of the mTOR pathway resulted into in Tg26 mice down regulation of renal tissue p53 expression, associated down stream signaling, and decreased number of sclerosed glomeruli, tubular microcysts, apoptosed, and 8-OHdG +ve cells in Tg26 mice. mTOR inhibition not only attenuated kidney cell expression of p66ShcA and phospho-p66ShcA but also reactivated redox-sensitive stress response program in the form of enhanced expression of MnSOD and catalase. In in vitro studies, the mTOR inhibitor also provided protection against HIV-induced podocyte apoptosis. Moreover, mTOR inhibition down regulated HIV-induced podocyte (HP/HIV) p53 expression. Since HP/HIV silenced for mTOR displayed lack of expression of p53 as well as attenuated podocyte apoptosis, this suggests that mTOR is critical for kidney cell p53 activation and associated oxidative kidney cell injury in HIV milieu.
- Identification of a highly conserved surface on Tat variants. [JOURNAL ARTICLE]
- J Biol Chem 2013 May 15.
Extra cellular Tat is suspected to protect HIV-1 infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the monoclonal antibody (mAb) 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized and its structure was stabilized with a disulfide bridge. Circular Dichroism spectra showed that MIMOOX had mainly beta turns but no alpha helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced condition. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine rational.
- HIV-2 and SIVmac accessory virulence factor Vpx down-regulates SAMHD1 catalysis prior to proteasome-dependent degradation. [JOURNAL ARTICLE]
- J Biol Chem 2013 May 15.
SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and SIV counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for poly-ubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C-terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits it catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.