Paramyxoviruses are the most important cause of respiratory disease in children. The long-term objective is to develop means to prevent respiratory disease caused by parainfluenza viruses. To reach that objective, the proposed research will focus on the roles of the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins during infection using two parainfluenza virus 1 subtypes: Sendai virus (SV), a natural pathogen of mice, and human parainfluenza type 1 (hPIV-1), an important pathogen of children. The strategy is to apply structure/function analysis to each of these viral proteins as outlined in the following specific aims.
Aim 1 : We propose to definitively establish a role of HN in virus fusion, structurally define the fusion promoting domain and the nature of the fusion promoting function.
Aim 2 : Determine the structural correlates and the biological significance of the neuraminidase activity of SV HN.
Aim 3 : To prepare molecules of HN and F that form crystals suitable for establishing the threedimensional structure.
Aims 1 and 2 will use epitope mapping, sequence analysis and functional characterization of neutralization escape mutants, ts mutants that fail to make HN, and expression of cDNAs in conjunction with site-directed mutagenesis to define the structural domains and biological significance of the fusion promoting and neuraminidase functions of HN. These methods will be used to define the antigenic structure, critical amino acids, and the extent and complexity of each domain. Studies using ts mutants and cDNA expression will examine the unresolved roles of the fusion promoting and neuraminidase activities of HN in virus infection. SV HN mutants with enhanced neuraminidase activity and significantly increased growth in embryonated eggs will be used to test the hypothesis that neuraminidase activity in general is a determinant of virus infectivity, pathogenesis and virulence.
Aim 3 will prepare F molecules with potential for crystallization and X-ray analysis. Requirements are (i) removal of the hydrophobic membrane anchoring termini (tails) by proteolysis, (ii) purification of tailless molecules by centrifugation and chromatography, and (iii) retention of biological activity. Antigen-MAb complexes will serve as an alternate method of crystallization.
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