The fusion of enveloped viruses with their target cells is directed by the viral transmembrane glycoprotein. The first part of this protein to interact with the cellular membrane is called the fusion domain and is a conserved, largely hydrophobic, polymorphic sequence usually near the amino terminus. Site-directed mutagenesis has shown that the replacement of key residues in the fusion domain of influenza virus hemagglutinin (HA2) or HIV glycoprotein 41,000 (gp41) affect viral fusion. Synthetic peptides with the same sequences as these N-terminal regions, termed fusion peptides (FP), induce lipid mixing and lysis of liposomes and cell membranes. Although there is much information on the structure of viral FP, there is little understanding of the relationship of the intramembrane FP structures to their function. The principal objective of the proposed research is to determine the structural characteristics common to viral FP that are necessary for fusion competence. Using FP based on mutated viral sequences, we will seek correlations between their altered activity and membrane-bound structures. We will also further characterize the inhibition of FP by the C-helix (DP-178) or its fragments and we will assess the ability of FP to expand its conformational space to include the formation of amyloid suprastructures. Membrane-perturbing activities of FP and its variants will be screened with erythrocyte lysis and aggregation measured by the absorbance of released hemoglobin at 540nm and cell sizing with a Coulter Counter. Lipid mixing, leakage, and aggregation of synthetic large unilamellar vesicles induced by FP will be measured using fluorescence dequenching and light scattering assays. The conformation, orientation, and topography of fusion peptides in membranes or membrane mimmicking solvents will be examined by circular dichroism (CD), Fourier transform infrared (FTIR), electron spin resonance (ESR) spectroscopy and molecular modeling. Correlations will be sought between the structural models and the fusion activities and lipid perturbations induced by viral fusion peptides and variants.
NIH ExPorter
NIH ExPorter