STRUCTURE-FUNCTION STUDY OF A HEPTAD REPEAT POSITIONED NEAR THE TRANSMEMBRANE DOMAIN OF SENDAI VIRUS FUSION PROTEIN WHICH BLOCKS VIRUS-CELLFUSION

A synthetic heptad repeat, SV-473, derived from Sendal virus fusion pr otein is a potent inhibitor of virus-cell fusion. In order to understa nd the mechanism of the inhibitory effect, we synthesized and fluoresc ently labeled SV-465, an extended version of SV-473 by one more heptad , its mutant peptide A(17,24)-SV-465, in which two heptadic leucines w ere substituted with two alanines, and its enatiomer D-SV-465, compose d entirely of D-amino acids. Similar mutations in the homologous fusio n protein of the Newcastle disease virus drastically reduced its activ ity. The data revealed that SV-465, but not A(17,24)-SV-465 or. its en antiomer, is highly active in inhibiting Sendai virus-induced hemolysi s of red blood cells. None of the peptides interfere with the binding of virions to the target red blood cells as demonstrated by hemaggluti nin assay. Fluorescence and circular dichroism (CD) spectroscopy indic ated that: (i) only SV-465 could self-assemble in aqueous environment; (ii) only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein; (iii) SV-465 has a higher helical content than A(17,24)-SV-465 in solution, and (i v) all the peptides bind strongly to zwitterionic and negatively charg ed phospholipids. Polarized attenuated total reflection infrared spect roscopy revealed that they bound as monomers onto the surface of zwitt erionic membranes with predominantly alpha-helical structures. The fun ctional role of the amino acid 465-497 domain in Sendai virus-mediated membrane fusion is discussed in light of these findings.