Book Description
The target of neutralizing antibodies (Abs) and entry inhibitors against HIV-1 is the envelope glycoprotein (Env) spike, a trimer of non-covalently associated gp120-gp41 heterodimers which mediates entry into target cells. The elicitation of neutralizing Abs against Env has been complicated by its instability and heterogeneity. To dissect Env heterogeneity on HIV-1, we developed a virus capture assay to probe virion-associated Env. We discovered that irrelevant Env varies greatly by genotype and expression vector, that immature gp160 persists at the virion surface, and that exogenous gp41 can bind to virions. We also assessed the functional stability of Env after exposure to heat, denaturants and prolonged incubations. Heat stability of Env trimers varied between HIV-1 isolates within a constrained range and correlated with stability to other conditions. The above studies revealed a complex picture of the antigenic surface of HIV-1. We next engineered Env trimers that were more stable and homogeneous than is typical. In the first approach, directed evolution of HIV-1 was used to select for virions displaying hyperstable Env trimers, and the stable Env was also more homogeneously trimeric than wild-type. These Env trimers will make interesting immunogens that do not require artificial stabilization. Second, chemical crosslinkers were used to covalently lock Env in trimeric conformation(s), after which irrelevant Env was depleted and Env trimers were purified. Immunization involving crosslinked Env trimers on proteoliposomes revealed that although crosslinking diminished the overall immunogenicity of Env, the Abs that were elicited sporadically neutralized different strains of HIV-1. Finally, we determined mechanistic details of a novel HIV-1 entry inhibitor, PF-68742. This compound blocks Env-mediated fusion at a post-attachment step. Env mutagenesis studies point to a gp120-gp41 interface involving gp120 C5, the disulfide loop and fusion peptide (FP) of gp41 as its likely target. PF-68742 and the inhibitor VIRIP both strongly enhanced the infectivity of viruses containing escape mutations in the FP. We hypothesize that these two inhibitors control FP insertion into the membrane by different mechanisms. Our results have illuminated new structure-function relationships in HIV-1 Env, and have produced new leads for the design of vaccine candidates and entry inhibitors against HIV-1.