Implications of HIV-1 Nef for “Shock and Kill” Strategies to Eliminate Latent Viral Reservoirs

Finding a cure for HIV is challenging because the virus is able to integrate itself into the host cell genome and establish a silent state, called latency, allowing it to evade antiviral drugs and the immune system. Various “shock and kill” strategies are being explored in attempts to eliminate latent HIV reservoirs. The goal of these approaches is to reactivate latent viruses (“shock”), thereby exposing them to clearance by viral cytopathic effects or immune-mediated responses (“kill”). To date, there has been limited clinical success using these methods. In this review, we highlight various functions of the HIV accessory protein Nef and discuss their double-edged effects that may contribute to the limited effectiveness of current “shock and kill” methods to eradicate latent HIV reservoirs in treated individuals

No evidence for selection of HIV-1 with enhanced Gag-Protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

Relative Resistance of HLA-B to Downregulation by Naturally Occurring HIV-1 Nef Sequences

ABSTRACT HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8+ T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef’s downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B. Here, we examined 46 Nef clones isolated from chronically HIV-1 subtype B-infected subjects for their ability to downregulate various HLA-A, HLA-B, and HLA-C molecules on the surface of virus-infected cells. Overall, HLA-B exhibited greater resistance to Nef-mediated downregulation than HLA-A, regardless of the cell type examined. As expected, no Nef clone downregulated HLA-C. Importantly, the differential abilities of patient-derived Nef clones to downregulate HLA-A and HLA-B correlated inversely with the sensitivities of HIV-infected target cells to recognition by effector cells expressing an HIV-1 Gag-specific T cell receptor. Nef codon function analysis implicated amino acid variation at position 202 (Nef-202) in differentially affecting the ability to downregulate HLA-A and HLA-B, an observation that was subsequently confirmed by experiments using Nef mutants constructed by site-directed mutagenesis. The in silico and mutagenesis analyses further suggested that Nef-202 may interact with the C-terminal Cys-Lys-Val residues of HLA-A, which are absent in HLA-B. Taken together, the results show that natural polymorphisms within Nef modulate its interaction with natural polymorphisms in the HLA cytoplasmic tails, thereby affecting the efficiency of HLA downregulation and consequent recognition by HIV-specific T cells. These results thus extend our understanding of this complex pathway of retroviral immune evasion

No evidence for selection of HIV-1 with enhanced gag-protease or nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

CAPRISA, 2013.Background: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and Results: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein down regulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. Conclusion: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 subtype C infection: association with disease progression and influence of immune pressure.

CAPRISA, 2014.Abstract available in pdf

Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity

HIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. We characterize SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones display lower ability to downregulate SERINC5 (median 80% activity) compared with progressor-derived clones (median 96% activity) (p = 0.0005). We identify 18 Nef polymorphisms associated with differential function, including two CTL escape mutations that contribute to lower SERINC5 downregulation: K94E, driven by HLA-B( *)08, and H116N, driven by the protective allele HLA-B( *)57. HIV-1 strains encoding Nef K94E and/or H116N display lower infectivity and replication capacity in the presence of SERINC5. Our results demonstrate that natural polymorphisms in HIV-1 Nef can impair its ability to internalize SERINC5, indicating that variation in this recently described function may contribute to differences in viral pathogenesis

Role of UDP-Glucuronic Acid Decarboxylase in Xylan Biosynthesis in Arabidopsis

UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell-wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDPglucuronic acid decarboxylase (UXS). Until now, little has been known about the physiological roles of UXS in plants. Here, we report that AtUXS1, AtUXS2, and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5, and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1 usx2 uxs4 triple mutant show no obvious phenotype, the uxs3 uxs5 uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in uxs3 uxs5 uxs6 and linkage analysis confirmed that the xylan content in uxs3 xus5 uxs6 declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel-permeation chromatography showed that the molecular weight of non-cellulosic polysaccharides in the triple mutants, mainly composed of xylans, is lower than that in the wild type, suggesting an effect on the elongation of the xylan backbone. Upon saccharification treatment stems of the uxs3 uxs5 uxs6 triple mutants released monosaccharides with a higher efficiency than those of the wild type. Taken together, our results indicate that the cytosol UXS plays a more important role than the Golgi-localized UXS in xylan biosynthesis

Impaired Nef Function Is Associated with Early Control of HIV-1 Viremia

UnlabelledHost and viral factors influence the HIV-1 infection course. Reduced Nef function has been observed in HIV-1 controllers during the chronic phase, but the kinetics and mechanisms of Nef attenuation in such individuals remain unclear. We examined plasma RNA-derived Nef clones from 10 recently infected individuals who subsequently suppressed viremia to less than 2,000 RNA copies/ml within 1 year postinfection (acute controllers) and 50 recently infected individuals who did not control viremia (acute progressors). Nef clones from acute controllers displayed a lesser ability to downregulate CD4 and HLA class I from the cell surface and a reduced ability to enhance virion infectivity compared to those from acute progressors (all P<0.01). HLA class I downregulation activity correlated inversely with days postinfection (Spearman's R=-0.85, P=0.004) and positively with baseline plasma viral load (Spearman's R=0.81, P=0.007) in acute controllers but not in acute progressors. Nef polymorphisms associated with functional changes over time were identified in follow-up samples from six controllers. For one such individual, mutational analyses indicated that four polymorphisms selected by HLA-A*31 and B*37 acted in combination to reduce Nef steady-state protein levels and HLA class I downregulation activity. Our results demonstrate that relative control of initial HIV-1 viremia is associated with Nef clones that display reduced function, which in turn may influence the course of HIV-1 infection. Transmission of impaired Nef sequences likely contributed in part to this observation; however, accumulation of HLA-associated polymorphisms in Nef that impair function also suggests that CD8+ T-cell pressures play a role in this phenomenon.ImportanceRare individuals can spontaneously control HIV-1 viremia in the absence of antiretroviral treatment. Understanding the host and viral factors that contribute to the controller phenotype may identify new strategies to design effective vaccines or therapeutics. The HIV-1 Nef protein enhances viral pathogenesis through multiple mechanisms. We examined the function of plasma HIV-1 RNA-derived Nef clones isolated from 10 recently infected individuals who subsequently controlled HIV viremia compared to the function of those from 50 individuals who failed to control viremia. Our results demonstrate that early Nef clones from HIV controllers displayed lower HLA class I and CD4 downregulation activity, as well as a reduced ability to enhance virion infectivity. The accumulation of HLA-associated polymorphisms in Nef during the first year postinfection was associated with impaired protein function in some controllers. This report highlights the potential for host immune responses to modulate HIV pathogenicity and disease outcome by targeting cytotoxic T lymphocyte (CTL) epitopes in Nef