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.

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).