The human immunodeficiency virus (HIV) gains entry to cells through the binding and membrane fusion activities of its envelope glycoprotein (Env). In an attempt to identify better targets for neutralizing antibodies and anti-HIV therapeutics, our research focusses on mapping sites critical to Env's binding and fusion activities. Over the past year I have established most of the pivotal assays and methods needed for our research objectives. This involved setting up a new lab, making and maintaining Env expression systems, setting up a fusion (activity) assay, securing reagents, etc. In addition, I've recruited lab personnel. Research projects have focussed on the mechanism and site of interaction of a specific peptide inhibitor of HIV. The peptide, obtained from Duke collaborator Dr. Carl Wild, is identical to a 38 residue sequence from the gp41 subunit of Env. In my fusion assay, I have shown that this peptide specifically inhibits Env-mediated membrane fusion and have characterized the inhibition kinetics. I have also shown that the peptide does not block Env binding to its receptor (CD4) and that the peptide preferentially binds Env-expressing cells (manuscript in preparation). Because my collaborator has shown that the peptide has a strong tendency to form a coiled-coil, and that mutations in the peptide that disrupt this structure also impair the peptide's HIV inhibitory activity, I am currently making a battery of Env mutations to test the coiled-coil hypothesis in Env's fusion activity. By inserting structure disrupting residues in the putative coiled-coil domain and testing their effect on the fusion activity , I hope to elucidate a possible role for a coiled-coil structure in the fusion activity of Env. Using a combinations of genetic and biochemical approaches, I also hope to identify structural and specificity requirements of the coiled-coil domain, which lies nascent to a principle neutralizing epitope. I also have initiated the makings of several reagents that I believe will be extremely valuable to future research. These include new Env expression systems and two mAbs.
