Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1.

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.

Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site.

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log(10) and 1.2 to 1.8 log(10) greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell.

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.

M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome.

Cytoplasmic transport of Gag molecules to the site of budding is an important but poorly understand process in retroviral assembly. Our previous studies of Mason-Pfizer monkey virus showed that, for this retrovirus, Gag is assembled into capsids at a pericentriolar region and that Env is necessary for efficient transport out of the site. An Env requirement for cytoplasmic transport implicates vesicular trafficking in this process even though the capsids remain cytoplasmic and do not bud into intracellular compartments in the cells studied to date. We show here that the secretory pathway of the cell is not directly involved in Gag transport since the latter was not inhibited by BFA, nor did Gag colocalize with markers of the ER, Golgi, or TGN. Instead, colocalization was observed between Gag and endocytosed transferrin and with Rab11, suggesting that pericentriolar recycling endosomes play a critical role in this process. Mutants of Rab11 that inhibit efflux of transferrin from the recycling endosome also inhibited Gag transport. Our studies show that Env colocalizes with Gag at the pericentriolar assembly site, and provide evidence that Env must travel through this compartment in order to initiate export of the capsids from the site of assembly. Thus, for the first time, endocytic trafficking of a retroviral Env glycoprotein is linked to the efficient cytoplasmic transport of Gag.

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics.

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.