The paramyxoviruses are the etiological agents of many important diseases of man and lower animals. Collectively, these viruses contribute significantly to worldwide morbidity and mortality. The focus of this study is the mechanism by which a prototype paramyxovirus, simian parainfluenza virus 5 (SV5) enters cells. The entry of enveloped viruses into cells requires the fusion of the viral envelope with a cellular membrane. The mechanism of viral-mediated membrane fusion is a topic of interest to cell biologists and structural biologists as well as those investigating the mechanism of virus entry. This is because membrane fusion is a process central in cell biology as it occurs at many stages of endocytosis and exocytosis. In addition, studies on the mechanism by which paramyxoviruses cause cell fusion are of significance for understanding the pathogenesis of human immunodeficiency virus 1. While our long-term goal is to use X-ray structural analysis to understand the molecular basis for paramyxovirus-mediated membrane fusion, we also need to use other functional approaches to identify determinants of paramyxovirus glycoproteins that are important for viral entry. We will study properties of the SV5 fusion (F) protein in six interrelated specific aims. We will analyze structure/function relationships of the SV5 F protein core trimer, which is composed of the F protein N-1 and C-1 heptad repeat regions, based on the atomic structure that we determined recently at 1.4 A resolution. We will investigate: (a) mutants that are predicted to alter core trimer stability; (b) mutants designed to investigate F protein conformation and (C) mutants designed to test properties of the fusion peptide. We will determine the mechanism of action of peptide inhibitors of SV5 fusion including: (a) determination of IC50 of peptide C-1 containing """"""""guest site"""""""" mutations at L447 and I449 which insert into a deep cavity in the N-1 trimer and an IC50/Tm ratio; (b) determination of the mechanism of peptide C-1 inhibition and capture of an early fusion-active conformation of SV5 F protein and (c) determination of the mechanism of peptide N-1 inhibition. We will investigate if core trimer formation brings the bilayers together for fusion and we will endeavor to detect F protein conformational change(s) required for fusion including conformational changes in F on cleavage from F0 to F1 + F2 and conformational changes in membrane bound and soluble forms of F1 + F2. We will investigate the roles of the F cytoplasmic tail and transmembrane domain on fusion activity and lipid 'raft' association. We will elucidate the role of specific mutations in the F protein in the context of a virus infection by using reverse genetics and we will study fusion activity, virus growth and virus assembly of the altered viruses.
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