Epistatic pathways can drive HIV-1 escape from integrase strand transfer inhibitors.

People living with human immunodeficiency virus (HIV) receiving integrase strand transfer inhibitors (INSTIs) have been reported to experience virological failure in the absence of resistance mutations in integrase. To elucidate INSTI resistance mechanisms, we propagated HIV-1 in the presence of escalating concentrations of the INSTI dolutegravir. HIV-1 became resistant to dolutegravir by sequentially acquiring mutations in the envelope glycoprotein (Env) and the nucleocapsid protein. The selected Env mutations enhance the ability of the virus to spread via cell-cell transfer, thereby increasing the multiplicity of infection (MOI). While the selected Env mutations confer broad resistance to multiple classes of antiretrovirals, the fold resistance is ~2 logs higher for INSTIs than for other classes of drugs. We demonstrate that INSTIs are more readily overwhelmed by high MOI than other classes of antiretrovirals. Our findings advance the understanding of how HIV-1 can evolve resistance to antiretrovirals, including the potent INSTIs, in the absence of drug-target gene mutations.

Neutral sphingomyelinase 2 is required for HIV-1 maturation.

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Impaired protective role of HLA-B*57:01/58:01 in HIV-1 CRF01_AE infection: a cohort study in Vietnam.

OBJECTIVES: Human Leukocyte Antigen HLA-B*57:01 and B*58:01 are considered anti-HIV-1 protective alleles. HLA-B*57:01/58:01-restricted HIV-1 Gag TW10 (TSTLQEQIGW, Gag residues 240-249) epitope-specific CD8+ T cell responses that frequently select for a Gag escape mutation, T242N, with viral fitness cost are crucial for HIV-1 control. Although this finding has been observed in cohorts where HIV-1 subtype B or C predominates, the protective impact of HLA-B*57:01/58:01 has not been reported in Southeast Asian countries where HIV-1 CRF01_AE is the major circulating strain. Here, the effect of HLA-B*57:01/58:01 on CRF01_AE infection was investigated. METHODS: The correlation of HLA-B*57:01/58:01 with viral load and CD4 counts were analyzed in the CRF01_AE-infected Vietnamese cohort (N = 280). The impact of the T242N mutation on CRF01_AE replication capacity was assessed. RESULTS: HLA-B*57:01/58:01-positive individuals mostly had HIV-1 with T242N (62/63) but showed neither a significant reduction in viral load nor increased CD4 counts relative to B*57:01/58:01-negative participants. In vitro and in vivo analyses revealed a significant reduction in viral fitness of CRF01_AE with T242N. In silico analysis indicated reduced presentation of epitopes in the context of CRF01_AE compared to subtype B or C in 10/16 HLA-B*57:01/58:01-restricted HIV-1 epitopes. CONCLUSION: The protective impact of HLA-B*57:01/58:01 on CRF01_AE infection is impaired despite strong suppressive pressure by TW10-specific CD8+ T cells.

Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations.

Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1-infected patients in the absence of mutations in drug target genes. We previously reported that, in vitro, the lab-adapted HIV-1 NL4-3 strain can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance viral cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can reduce susceptibility to multiple classes of ARVs and also increase resistance to ARVs when coupled with target-gene mutations. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation and lower rates of gp120 shedding than the WT virus. We also selected for Env mutations in clinically relevant HIV-1 isolates in the presence of ARVs. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication and Env structure that are HIV-1 strain dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest that mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route may increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV target genes.IMPORTANCE Although combination antiretroviral (ARV) therapy is highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle. Recent findings suggest that resistance can develop without ARV target gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here, we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo We demonstrate that Env mutations can reduce sensitivity to major classes of ARVs in multiple viral isolates and define the effect of the Env mutations on Env subunit interactions. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based Env-mediated drug resistance may impact therapeutic strategies and provide clues toward understanding how ARV-treated individuals fail therapy without acquiring mutations in drug target genes.

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation.

The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation.IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.

HIV-1 matrix mutations that alter gag membrane binding modulate mature core formation and post-entry events.

The matrix (MA) domain of HIV-1 Gag directs membrane binding of the Gag precursor polyprotein during the late events of virus replication. However, the effects of alteration in Gag membrane binding early post-infection are not well understood. To investigate impacts of MA mutations that alter Gag membrane binding on the phenotypes of newly produced virus particles, we extensively characterized two MA mutants by virological, biochemical, and morphological approaches. The V6R mutation, which decreases Gag membrane binding, modified Gag processing and core morphogenesis and impaired core uncoating, reverse transcription, and viral DNA integration. On the other hand, the L20K mutation, which increases Gag membrane binding, primarily decreased integrated DNA levels without affecting the viral components and morphology. These data suggest that HIV-1 MA plays roles in functional core formation and the following post-entry steps of the virus replication cycle. (140/150 words).

Increased HIV-1 sensitivity to neutralizing antibodies by mutations in the Env V3-coding region for resistance to CXCR4 antagonists.

HIV-1 passage in cell culture in the presence of chemokine receptor antagonists can result in selection of viruses with env mutations that confer resistance to these inhibitors. In the present study, we examined the effect of HIV-1env mutations that confer resistance to CXCR4 antagonists on envelope (Env) sensitivity to neutralizing antibodies (NAbs). Serial passage of CXCR4-tropic HIV-1 NL4-3 in PM1/CCR5 cells under CXCR4 antagonists KRH-3955, AMD3100 and AMD070 yielded two KRH-3955-resistant, one AMD3100-resistant and one AMD070-resistant viruses. These viruses had multiple env mutations including the Env gp120 V3 region. The majority of viruses having these CXCR4 antagonist-resistant Envs showed higher sensitivity to NAbs 447-52D, b12 and 2F5 targeting the V3 region, the gp120 CD4-binding site and the gp41 membrane proximal region, respectively, compared to NL4-3 WT virus. Recombinant NL4-3 viruses with the V3-coding region replaced with those derived from the CXCR4 antagonist-resistant viruses showed increased sensitivity to NAbs b12, 2F5 and 447-52D. Molecular dynamics simulations of Env gp120 outer domains predicted that the V3 mutations increased levels of fluctuations at the tip and stem of the V3 loop. These results indicate that mutations in the V3-coding region that result in loss of viral sensitivity to CXCR4 antagonists increase viral sensitivity to NAbs, providing insights into our understanding of the interplay of viral Env accessibility to chemokine receptors and sensitivity to NAbs.