Dissecting the mechanism of viral RNA recombination

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DESCRIPTION (provided by applicant): RNA recombination contributes greatly to RNA virus evolution via promoting emergence of new pathogenic or drug-resistant viruses and strains. Another important function of RNA recombination is to repair damaged or mutated viral genomes, contributing to virus fitness. RNA recombination can also occur between viral and host sequences, leading to the emergence of recombinant viruses having novel traits. RNA recombination has been demonstrated to occur for a large number of human viruses, including Picorna-, Flavi-, Rota-, Corona-, Calici-, Arteri-, Astro-, Alphaviruses, hepatitis D virus and influenza A virus. In spite of its significance, understanding of the mechanism of RNA recombination is incomplete. The PI has developed efficient and tractable in vitro RNA recombination systems based on purified Tombus- and Carmovirus RNA-dependent RNA polymerases (RdRp), which are similar to the RdRp of a number of significant human viruses. The PI has also developed an efficient in vivo RNA recombination system in whole plants and single cells (protoplasts), which make Tombusviruses exceptionally suitable for studying RNA recombination. The central hypothesis tested in this work is that there are specific RNA recombination signals in the recombining RNAs and these signals can be recognized by the viral RdRp. Encouraged by preliminary data, the PI will dissect (Aim 1) the mechanism of RNA recombination supported by (i) replication enhancers; (ii) AU-rich sequences; and (iii) highly structured sequences using the Tombus- and Carmovirus RdRp systems; and he will test (Aim 2) recombination-promoting sequences in single cells and in whole organisms in order to estimate their efficiencies in RNA recombination. The possible significance of these elements in interviral recombination will be examined by testing the ability of a Carmovirus replication enhancer to support RNA recombination in the Tombusvirus system. The research described here will lead to better understanding of the mechanism of RNA recombination and the interaction between replication elements and the viral RdRp during RNA recombination. Accomplishing the proposed goals will provide crucial information towards identification of universal recombination signals and will help developing computer software to predict recombination and virus evolution. Knowledge of recombination promises untold health benefits by providing means to limit virus infections and safer vaccines.