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 strategyone 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.
Root, M.J., M.S. Kay and P.S. Kim. Protein Design of an HIV-1 Entry Inhibitor. Science. 291: 884-888, 2001.
Root, M.J. and D.H. Hamer. Targeting therapeutics to an exposed and conserved binding element of the HIV-1 fusion protein. Proceedings of the National Academy of Sciences, U.S.A. 100: 5016-5021, 2003.
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