The aim of the proposed research is to contribute to a more detailed molecular description of the poliovirus neutralizing sites and the immune response induced by the virus. A combination of genetic and immunological approaches are proposed to test predictions derived from a structural analysis of the neutralizing sites, thereby A) extending the structural description of the previously identified neutralizing sites for poliovirus serotype 1, B) identifying potentially new neutralizing sites, and C) functionally characterizing regions in the capsid proteins that are critical for the viability of the virus. To achieve these goals, several studies are proposed: 1) Viral mutants will be constructed using site-specific mutagenesis methods. The molecular biology and antigenic characteristics of these mutants will be studied. 2) Regions within the capsid proteins that appear to be hypermutable or mutationally constrained will be identified by sequencing six naturally-occuring poliovirus serotype 1 strains. These strains were sequentially isolated over a 13 month period by the Centers for Disease Control from cases that occurred during an epidemic outbreak of paralytic poliomyelitis within the Amish community. This study will indicate the spectrum of amino acid substitutions and the regions within the virus that viable mutations can occur. 3) The immunogenicity of the VP4 capsid protein will be studied. Although the immunogenicity of the other three capsid proteins have been extensively studied, the antibody response to VP4 is uncharacterized and may play important roles in poliovirus pathogenesis. The studies proposed here provide an unique opportunity to extend our understanding beyond the identification of the amino acids that form a neutralizing determinant and to address issues concerning: how antibodies interact with the neutralizing sites, what functional roles do different regions on the virus surface (including the neutralizing sites) serve for the biology of the virus, how viruses mutate to become neutralization resistant and generate antigenic variants, how are new serotypes formed. These issues are important for a thorough understanding of the molecular mechanisms of viral pathogenesis. In addition, the health relatedness of this proposal derives from its contribution to the understanding the molecular basis of host protection by viral vaccines and the potential application of this knowledge to the rational design of vaccines for viruses (e.g. HTLV and HIV strains, and hepatitis A) and for the improvement of existing viral vaccine (such as poliovirus).
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