My laboratory studies the mechanism of HIV entry and its inhibition. HIV cellular invasion begins with fusion of the viral and cellular membranes, a process mediated by the viral envelope glycoprotein complex gp120/gp41. The interaction of gp120 with cellular receptors (CD4 and a chemokine coreceptor) triggers gp41 to extend and grab hold of the target cell membrane. This transient, extended (“prehairpin”) intermediate subsequently collapses into a trimer-of-hairpins structure that brings the amino- and carboxyl-terminal regions of the gp41 extracellular domain into close proximity. In doing so, the membranes are brought together in a manner that facilitates membrane fusion. Peptides derived from the carboxyl-terminal region (called C-peptides) can inhibit membrane fusion by binding to the gp41 amino-terminal region and disrupting trimer-of-hairpins formation. Recently, we designed a small protein, called 5-Helix, to test a converse inhibitory strategy—one that targets the C-peptide region of gp41. 5-Helix is a monomeric protein designed to contain most of the gp41 trimer-of-hairpins except that one C-peptide region is absent. This vacancy creates a very high affinity-binding site for the carboxyl-terminal region of the gp41 ectodomain. 5-Helix displays potent (nanomolar) inhibitory activity against a diverse set of HIV-1 variants, validating the C-peptide region of gp41 as a viable target for membrane fusion inhibition.

Currently, we are probing the inhibitory mechanisms of 5-Helix and C-peptides in hopes of discovering better strategies to prevent HIV membrane fusion. We are studying the interaction of 5-Helix with C-peptides in detail in order to address some of the fundamental questions regarding their fusion inhibition. Are the inhibitions by C-peptides and 5-Helix thermodynamically driven, or do they represent a kinetic or catalytic process? How many C-peptides/5-Helix molecules per gp41 trimer are required to inhibit membrane fusion? Can we rationally design C-peptides or 5-Helix proteins with enhanced inhibitory activities? We are using variants of 5-Helix to probe the exposure and accessibility of the C-peptide region of gp41 during the various stages of the fusion process. And through proteomic and genetic techniques, we are searching for small molecules and peptides that interact with the C-peptide binding site of 5-Helix and display inhibitory activity.

In addition, we plan to test the generalizability of the 5-Helix design strategy on inhibiting viral entry. Many enveloped viruses, including Ebola virus, respiratory syncytial virus and human T-cell leukemia virus (HTLV), utilize a trimer-of-hairpins motif to facilitate membrane fusion. For some of these viruses, 5-Helix variants based upon these trimer-of-hairpin structures might display similar antiviral activity as the HIV 5-Helix. These studies will enhance our general understanding of viral membrane fusion and provide leads into designing new inhibitory agents for these viral illnesses.

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