This proposal describes the use of recombinant DNA technology for the study of poliovirus, an RNA virus. Recently it was found that a complete cloned cDNA copy of the poliovirus genome is infectious in mammalian cells. The infectious cDNA clone will be used to perform genetic manipulations with poliovirus which were not previously possible. Initially two general methods for employing the cDNA clone to study poliovirus will be developed, and these methods will be used to address several specific problems in poliovirus replication. One method for performing genetic studies will be to specifically mutagenize the cDNA, introduce the altered cDNA into cultured mammalian cells, and observe the phenotype. To employ this approach we will search for ways of increasing the specific infectivity of the cDNA clone so that transfectants may be examined biochemically. A second general method involves the construction of """"""""permissive"""""""" cell lines which produce poliovirus proteins. These cell lines may be used to propagate mutant viruses into which deletions have been inserted by manipulation of the infectious cDNA. These two general methodologies will be used to study aspects of poliovirus replication which include (1) the mechanism by which the cDNA clone is infectious in mammalian cells; (2) the study of defective interfering particles through introduction of specific deletions in the capsid region; (3) the ability of cell lines producing poliovirus proteins to complement poliovirus mutants, and (4) the function of protein NCVPX. The results will contribute to our goal of obtaining a complete description of the replication of a human pathogen.
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