Direct evidence that the N-terminal heptad repeat of sendai virus fusion protein participates in membrane fusion

Recent studies have demonstrated the importance of heptad repeat regions wi thin envelope proteins of viruses in mediating conformational changes at va rious stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, f luorescently labeled and functionally and structurally characterized a wild -type 70 residue peptide (SV-117) composed of both the fusion peptide and t he N-terminal heptad repeat of Sendai virus fusion protein, two of its muta nts, as well as the fusion peptide and heptad repeat separately. One mutati on was introduced in the fusion peptide (G119K) and another in the heptad r epeat region (I154K). Similar mutations have been shown to drastically redu ce the fusogenic ability of the homologous fusion protein of Newcastle dise ase virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidyl-choline/phosphatidyiglycerol (PC/PG) large unilamellar ve sicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and t o a lesser extent its two mutants, were potent inhibitors of Sendai virus-m ediated hemolysis of red blood cells, while the fusion peptide and SV-150 w ere negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells . Further studies revealed that SV-117 and its mutants oligomerize similarl y in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher he lical content for SV-117 compared to its mutants in 40% tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated t hat SV-117 lies more parallel with the surface of the membrane than its mut ants. These observations suggest a direct role for the N-terminal heptad re peat in assisting the fusion peptide in mediating membrane fusion. (C) 1999 Academic Press.