This project will continue quantitative analysis of the great mutational variability of RNA viruses, and of their very rapid evolution or paradoxical genetic stability during replication. The rapid rates of evolution of many RNA viruses (such as HIV (AIDS) viruses, influenza viruses, poliovirus vaccine strains, etc.) are extensively documented, but basic molecular mechanisms and population dynamics remain poorly understood. Newly-developed methods for quantitating base substitution error frequencies of viral RNA polymerase will be improved to increase sensitivity and accuracy, and will be applied to many defined single base sites on genomes of vesicular stomatitis virus (VSV) and other virus (and cellular) RNAs. This will determine relative constancy or variability of polymerase error rates at many sites, and generally quantitate the """"""""quasispecies"""""""" (heterogenous) nature of populations of RNA viruses (and RNA molecules) from various backgrounds. The roles of relative growth rates, """"""""founder effects"""""""", and competitive abilities in shaping the population dynamics of RNA virus populations will be examined in reconstruction experiments employing clones of defined single base mutants and clones of """"""""wild type consensus sequence"""""""" viruses. Studies of immune system selection of VSV mutants will be continued to provide detailed information regarding VSV glycoprotein (and other protein) epitopes involved in escape from monoclonal antibodies and from natural killer cell destruction of persistently-infected tumors in nude mice. The monoclonal antibody escape studies will also aid in analysis of RNA virus genetics and population structure. Studies of the rapid coevolution of virus and defective interfering (DI) particles will examine the molecular basis for rapidly- changing viral replicase specificities. This will be characterized with cell-free VSV replication systems, and by sequencing of the L protein polymerase genes and of the viral and DI termini for which they exhibit changing specificities. These viral and DI particle mutants will also be employed in attempts to detect recombination of viable virus genomes (not yet observed for negative strand RNA viruses). Finally cell-free replication systems will be employed in attempts to generate new DI particles and to explore in vitro the polymerase template-switching-""""""""copyback"""""""" mechanism for RNA recombination.
Showing the most recent 10 out of 46 publications
