Abstract
A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion. In order to understand the mechanism of the inhibitory effect, we synthesized and fluorescently labeled SV-465, an extended version of SV-473 by one more heptad, its mutant peptide A17,24-SV-465, in which two heptadic leucines were substituted with two alanines, and its enatiomer D-SV-465, composed entirely of Damino acids. Similar mutations in the homologous fusion protein of the Newcastle disease virus drastically reduced its activity. The data revealed that SV-465, but not A17,24-SV-465 or its enantiomer, is highly active in inhibiting Sendai virus-induced hemolysis of red blood cells. None of the peptides interfere with the binding of virions to the target red blood cells as demonstrated by hemagglutinin assay. Fluorescence and circular dichroism (CD) spectroscopy indicated 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 A17,24-SV-465 in solution, and (iv) all the peptides bind strongly to zwitterionic and negatively charged phospholipids. Polarized attenuated total reflection infrared spectroscopy revealed that they bound as monomers onto the surface of zwitterionic membranes with predominantly alpha-helical structures. The functional role of the amino acid 465-497 domain in Sendai virus-mediated membrane fusion is discussed in light of these findings.
Highlights
A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion
The results show that SV-465 is more potent than SV-473 in inhibiting Sendai virus-mediated hemolysis, whereas both the mutant A17,24-SV-465 and the enantiomer D-SV-465 are inactive
In order to check whether SV-465 can exhibit its inhibition potential when the virions are already bound to red blood cells (RBCs), Sendai virions were first added to RBCs and incubated for 30 min to permit viral attachment, and SV-465 was added (Fig. 2, top panel, column D)
Summary
Materials—BOC-protected phenylacetamidomethyl resins and dimethylformamide (peptide synthesis grade) were purchased from Applied Biosystems (Foster City, CA), and BOC amino acids were obtained from Peninsula Laboratories (Belmont, CA). NBD and Rhodamine Fluorescence Measurements—Fluorescence emission spectra of NBD- and Rho-labeled peptides either in PBS or in the presence of lipid vesicles were recorded at room temperature on a Perkin-Elmer LS-50B spectrofluorometer, with the excitation monochromator set at 467 and 530 nm, respectively, with a 5– 8 nm slit width. The correction for light scattering was made by subtracting the signal obtained when unlabeled peptides, at concentrations equal to the sum of the donor and the acceptor, were added to vesicles. Samples were prepared as described previously [54], briefly, a mixture of PC (0.7 mg) alone or with a peptide (lipid/peptide molar ratio 55:1) was deposited on a Germanium prism (52 ϫ 20 ϫ 2 mm). The electric field components together with the dichroic ratio (defined as the ratio between absorption of parallel (to a membrane plane), Ap, and perpendicularly polarized incident light, As) are used to calculate the orientation order parameter, , by the following formula,
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
