BIOSYNTHESIS OF PARAMYXOVIRUSES

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Paramyxoviruses are the most important cause of respiratory disease in children. The long-term objectives of this project are to elucidate the structural, molecular, and biological determinants of paramyxovirus host tropism, virulence, and protective immunity using Sendai virus, an archetypal paramyxovirus. To this end the proposed studies are focused on the role of the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the virion. The specific aims are: (1) characterization of biologically active fragments of HN and F with potential for crystallization, (2) to locate on the primary amino acid sequence the epitopes that define the antigenic structure and functions of HN and F, and (3) to determine the influence of host cells and immune-pressure on the selection of Sendai virus populations with altered host range and virulence. To prepare biologically active fragments of HN and F with a possibility for crystallization and x-ray analyses, the hydrophobic terminus will be removed by proteolysis. The cleavage product will be purified by ion exchange chromatography and rate zonal centrifugation. Before attempting crystallization, the cleaved proteins will be examined for retention of biological activities; neuraminidase for HN and antibody binding for both HN and F. Amino acid sequence analysis will determine the precise point of cleavage. Crystallization of an antigen-monoclonal antibody complex will be attempted and served as an alternative method to produce analyzable crystals and as a way to analyze their molecular interaction. Second, monoclonal antibodies will be used to map the antigenic structure and determine the functional activities of HN and F. Nucleotide sequence analysis of antigenic variants selected with the monoclonal antibodies will localize antigenic and functional sites on the primary amino acid sequence. This information will be correlated with the structural information. Third, Sendai virus primary isolates will be grown in mammalian and avian cells. The viruses will be characterized with monoclonal antibodies to the surface glycoproteins to determine whether antigenically distinguishable viruses are selected in different host cells. Antigenic changes will be correlated with sequence changes in the HN and F molecules and alterations in the host tropism and virulence.