Ingrid Markovic Tuesday, Oct. 22, 2002 12:15pm Building 4, Room 433 Functional determinants of HIV-1 entry: Cellular factors involved in trimolecular complex restructuring leading to fusion Abstract: HIV entry into target cells requires attachment of the gp120 subunit of the viral envelope (Env) glycoprotein to its primary receptor, CD4. This interaction induces a structural rearrangement in the molecule exposing conserved regions of gp120, which enables binding to coreceptors, CXCR4 or CCR5. Ligation of gp120 to its coreceptor triggers a conformational change in the gp41 subunit, which leads to exposure and subsequent insertion of the gp41 fusion peptide into the target membrane, followed by fusion of two apposing membranes and release of the viral capsid into the cytosol. Our current work adds an additional layer of complexity to the established model of HIV entry by suggesting that catalysts with redox-isomerase activity such as protein disulfide isomerase (PDI), initially identified by Ryser et al. (PNAS, 91:4559, 1994), assists the Env to acquire its fusion-competent conformation enabling fusion of viral and cell membrane. We find that exogenously added PDI can rescue fusion from block imposed by anti PDI agents. Furthermore, at the contact site of viral and cell membranes gp120 acts as an assembly mechanism bringing PDI into complex with CD4 and CXCR4. To assess if gp120 undergoes disulfide-based isomerization enabling the protein to acquire new conformation we used biotin maleimide to label free thiol groups. Such thiol/disulfide shuffling requires reduction of existing, and formation of new disulfide bonds, pairing previously unpaired cysteines. Since no free thiols are present in the gp120 ectodomain, incorporation of biotin maleimide indicates reduction of one or multiple disulfide bonds implying that gp120 undergoes thiol/disulfide rearrangement during conformational change. Our findings are consistent with the assumption that PDI acts to effectively lower the energy barrier to Env conformational rearrangement much like low pH or elevated temperatures in the case of influenza viral hemagglutinin.

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