The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BackgroundThe p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy.ResultsRemarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV.ConclusionsOur results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor

Inhibition of HIV early replication by the p53 and its downstream gene p21

Abstract Background The tumor suppressor gene p53 has been found to suppress HIV infection by various mechanisms, but the inhibition of HIV at an early stage of replication by host cell p53 and its downstream gene p21 has not been well studied. Method VSV-G pseudotyped HIV-1 or HIV-2 viruses with GFP or luciferase reporter gene were used to infect HCT116 p53+/+ cells, HCT116 p53−/− cells and hMDMs. The infections were detected by flow cytometry or measured by luciferase assay. Reverse transcription products were quantified by a TaqMan real time PCR. siRNA knockdown experiments were applied to study potential roles of p53 and p21 genes in their restriction to HIV infection. Western blot experiments were used to analyze changes in gene expression. Results The infection of HIV-1 was inhibited in HCT116 p53+/+ cells in comparison to HCT116 p53−/− cells. The fold of inhibition was largely increased when cell cycle switched from cycling to non-cycling status. Further analysis showed that both p53 and p21 expressions were upregulated in non-cycling HCT116 p53+/+ cells and HIV-1 reverse transcription was subsequently inhibited. siRNA knockdown of either p53 or p21 rescued HIV-1 reverse transcription from the inhibition in non-cycling HCT116 p53+/+ cells. It was identified that the observed restrictions by p53 and p21 were associated with the suppression of RNR2 expression and phosphorylation of SAMHD1. These observations were confirmed by using siRNA knockdown experiments. In addition, p53 also inhibited HIV-2 infection in HCT116 p53+/+ cells and siRNA knockdown of p21 increased HIV-2 infection in hMDMs. Finally the expressions of p53 and p21 were found to be induced in hMDMs shortly after HIV-1 infection. Conclusions The p53 and its downstream gene p21 interfere with HIV early stage of replication in non-cycling cells and hMDMs

Long-term antiretroviral therapy mitigates mortality and morbidity independent of HIV tropism: 18 years follow-up in a women's cohort

ObjectiveCXCR4 (X4)-tropic HIV-1 was found previously to herald CD4 + cell depletion and disease progression in individuals who were antiretroviral-naive or took combination antiretroviral therapy (cART) for less than 5 years. We updated this finding by investigating whether the deleterious effect of X4-tropic strains is mitigated by long-term cART.DesignWe examined morbidity and mortality in relation to HIV-1 tropism and cART in 529 participants followed up to 18 years in the Women's Interagency HIV Study; 91% were women of color.MethodsPlasma-derived HIV-1 tropism was determined genotypically.ResultsWe categorized participants according to the number of visits reported on cART after initiation. Group 1: three or less visits, 74% of these participants reporting no cART; group 2: at least four visits and less than 70% of visits on cART; group 3: at least 70% of visits on cART. AIDS mortality rates for participants in each group with X4 virus compared with those with R5 virus exclusively were, respectively: 62 vs. 40% ( P  = 0.0088); 23% vs. 22% [nonsignificant (NS)]; 7% vs. 14% (NS). Kaplan-Meier curves showed accelerated progression to AIDS death or AIDS-defining illness in participants with three or less cART visits and X4 viruses ( P  = 0.0028) but no difference in progression rates stratified by tropism in other groups. Logistic regression found that HIV-1 suppression for at least 10 semiannual visits (≥5 years total) mitigated X4 tropism's deleterious effect on mortality, controlling for maximal viral load, and CD4 + nadir.ConclusionLong-term cART markedly mitigated the deleterious effect of X4 viruses on AIDS morbidity and mortality. Mitigation was correlated with duration of viral suppression, supporting HIV-1 suppression as a crucial goal

Long-Term Antiretroviral Therapy Mitigates Mortality and Morbidity Independent of HIV Tropism: 18 Years Follow-Up in a Women\u27s Cohort

OBJECTIVE: CXCR4 (X4)-tropic HIV-1 was found previously to herald CD4 + cell depletion and disease progression in individuals who were antiretroviral-naive or took combination antiretroviral therapy (cART) for less than 5 years. We updated this finding by investigating whether the deleterious effect of X4-tropic strains is mitigated by long-term cART. DESIGN: We examined morbidity and mortality in relation to HIV-1 tropism and cART in 529 participants followed up to 18 years in the Women\u27s Interagency HIV Study; 91% were women of color. METHODS: Plasma-derived HIV-1 tropism was determined genotypically. RESULTS: We categorized participants according to the number of visits reported on cART after initiation. Group 1: three or less visits, 74% of these participants reporting no cART; group 2: at least four visits and less than 70% of visits on cART; group 3: at least 70% of visits on cART. AIDS mortality rates for participants in each group with X4 virus compared with those with R5 virus exclusively were, respectively: 62 vs. 40% ( P  = 0.0088); 23% vs. 22% [nonsignificant (NS)]; 7% vs. 14% (NS). Kaplan-Meier curves showed accelerated progression to AIDS death or AIDS-defining illness in participants with three or less cART visits and X4 viruses ( P  = 0.0028) but no difference in progression rates stratified by tropism in other groups. Logistic regression found that HIV-1 suppression for at least 10 semiannual visits (≥5 years total) mitigated X4 tropism\u27s deleterious effect on mortality, controlling for maximal viral load, and CD4 + nadir. CONCLUSION: Long-term cART markedly mitigated the deleterious effect of X4 viruses on AIDS morbidity and mortality. Mitigation was correlated with duration of viral suppression, supporting HIV-1 suppression as a crucial goal

The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities

Abstract Background HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. Results Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and −2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. Conclusions The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways

SFN blocks infection after entry and but before 2-LTR circle formation.

<p>Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of <i>nef</i>. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).</p

SFN action impacts HIV-2 as well as HIV-1 and is not reporter dependent.

<p>PMA-differentiated THP1 cells were treated with media supplemented with vehicle only (DMSO), 5 μM AZT or with 10 μM SFN. Twenty-four hours after treatment, the samples were either mock infected or infected with (A), VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i> or (B), VSV-G pseudotyped HIV-2 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after infection, luciferase activity was measured by photon emission. (C), In parallel, the same experiment as in (A) and (B) was performed except that THP1 cells were infected with VSV-G-pseudotyped HIV-1 with GFP in place of <i>nef</i> or (D), VSV-G pseudotyped HIV-2 with GFP in place of <i>nef</i>. The samples with GFP-reporter viruses were fixed and harvested 24 h after infection and the fraction of GFP(+) cells was enumerated by flow cytometry. Bar graphs represent the data for replicate experiments (n = 3).</p

SFN action blocks spreading infections that rely on the HIV envelope for viral entry.

<p>hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. The bar graphs represent the data for replicate experiments (n = 3).</p

SFN does not trigger expression of interferon-stimulated anti-viral factors SAMHD1 or MX2.

<p>PMA-differentiated THP1 cells were mock-treated or treated with media supplemented with vehicle only (DMSO) or with 10 μM SFN or with 500 U/mL of IFNα. (A), Proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. (B), Densitometric analysis was performed on the Nrf2, SAMHD1, MX2, NQO1 and GCLM (NQO1 and GCLM are both indicators of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the bands were then graphed.</p