Kinetics of HIV-1 Fusion Inhibition: Implications for Viral Resistance and the Design of Improved Fusion Inhibitors
Abstract
HIV-1 entry into host cells is mediated by the trimeric envelope glycoprotein (Env). During the fusion process, two important regions of the gp41 transmembrane Env subunit are transiently exposed. HIV-1 fusion inhibitors C37 and 5-Helix respectively target these N- and C-terminal heptad repeat regions (N-HR and C-HR), blocking formation of the gp41 trimer-of-hairpins crucial to viral membrane fusion. The focus of this work was to develop a detailed understanding of the determinants of fusion inhibition, evolution of viral resistance to fusion inhibitors, and the fusogenic structural transitions in an effort to develop improved fusion inhibitors. In the first portion of these studies, C37 variants were generated and their inhibitory activities and binding parameters were analyzed to determine the biophysical properties that influence inhibition. C37 potency was shown to be related to binding affinity, in contrast to previous reports that 5-Helix inhibition depends upon inhibitor association kinetics. This discrepancy was attributed to a difference in the rate of irreversible deactivation of inhibitor-bound gp41: 5-Helix rapidly shuttles gp41 to a fusion-incompetent form, while C37 must remain bound substantially longer to deactivate gp41. Since C37 potency depends upon the inhibitor occupying gp41 for a sufficient period of time to promote deactivation, the impact of binding affinity on resistance acquisition was investigated. By generating viruses resistant to C37 and a higher-affinity variant, C37KYI, it was shown that increasing inhibitor affinity poses a greater barrier to resistance. An examination of the resistance profiles revealed that the majority of escape mutations cluster in the inhibitor binding site and disrupt affinity. Additionally, some distant mutations accelerate viral fusion to kinetically restrict inhibitor target access, while others appear to compensate for fitness defects attained during Env mutagenesis. Interestingly, highly-resistant viruses were potently inhibited by a dimeric C37 variant. In the final portion of these studies, heterotrimeric Env viruses were utilized to determine the stoichiometry of fusion inhibition. It was demonstrated that although the gp41 trimer has three identical binding sites, a single inhibitor is sufficient to prevent infection, implying that fusion requires the folding of all three gp41 subunits. Together, these studies suggest that accelerating inhibitor-mediated gp41 deactivation, enhancing inhibitor binding affinity, and increasing inhibitor avidity are attractive approaches for the design of improved HIV-1 fusion inhibitors.
Subject Area
Molecular biology|Pharmacology|Virology
Recommended Citation
Kahle, Kristen M, "Kinetics of HIV-1 Fusion Inhibition: Implications for Viral Resistance and the Design of Improved Fusion Inhibitors" (2009). ProQuest ETD Collection - Thomas Jefferson University. AAI3535987.
https://jdc.jefferson.edu/dissertations/AAI3535987
