The long-term goal of this research is to determine the molecular mechanisms of replication, a process fundamental to pathogenicity arid virulence, in (+)-strand RNA viruses that include many human, animal and plant pathogens. The use of powerful model eukaryotic virus systems, such as the turnip crinkle virus system, is necessary to address many important questions in RNA virus replication, and results have already been shown to be applicable to human viruses. TCV is associated with small, non-coding parasitic RNAs like satC that contain cis-acting elements recognized by the TCV RdRp. Already identified are: a 3-terminal carmovirus consensus sequence (CCS); 3PE also containing a CCS; the motif1-hairpin (M1 H) that is a recombination hot-spot, enhancer of transcription, and also contains a CCS; and 5 PE required for (+)-strand synthesis. The similar sequences that comprise the elements and the ability of the 3PE and 5PE to serve as independent promoters in vitro, suggests that they all attract the RdRp. To address the function and relationship among the elements, Specific Aim 1 will test the hypothesis that the in vivo and in vitro structures of satC (-)strands differ in the accessibility of the 3 end to the RdRp, with the in vitro structure promoting internal primer extension and the in vivo structure promoting full-length complementary strand synthesis. We will also convert the in vitro structure into the more active in vivo structure using mutations to stabilize the in vivo structure and RNA chaperones.
In Specific Aim 2, an analysis of the M1 H of satC and theanalogous motif-3 hairpin of TCV will be conducted. We will test the hypothesis that functional replacement sequences can enhance either replication of satC or systemic movement of TCV. We will also test why the most common Ml H replacement motif CAACCCC, also found in the 5PE of the related virus Cardamine chlorotic fleck (CCFV), inhibits replication in certain sequence contexts. Loop-out and scanning models will be tested for how the RdRp finds the 3 end promoter after M1H binding.
In Specific Aim 3, a functional analysis of the 5PE of satC, TCV and CCFV will be conducted, addressing the sequence specific nature of the 5 PE and determining whether the element, which can serve as an independent promoter for the TCV RdRp in vitro, plays an enhancer or a structural role in vivo.
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