Initiation of (-)-Strand Synthesis in Turnip Crinkle Virus Associated RNAs

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0086952 Anne SimonAlthough RNA viruses have been the subjects of intensive investigations for the past 100 years, replication, a fundamental process in the life cycle of a virus, remains poorly understood. The viral-encoded enzyme involved in replication, the RNA-dependent RNA polymerase (RdRp), together with possible proteins from the host, must recognize specific viral RNAs, initiate complementary strand synthesis and then use these complementary strands as templates for synthesis of the viral genome. Promoters for RdRp are poorly understood, and much of the research to date involves viruses terminating with either tRNA-like structures (e.g., Brome mosaic virus) or poly(A) tails (e.g., Poliovirus). Much less is known about viruses whose genomic RNA(s) terminate with a free hydroxyl group such as turnip crinkle virus (TCV). In addition, it is only recently becoming clear that internal sequences, and not just end sequences, are important for initiation of transcription by RdRp. Possible roles for such sequences are (i) RdRp-binding sequences, or (ii) sequences that engage in RNA-RNA interactions with end sequences to bind the RdRp to the viral genome. TCV is used as a model to study sequences and structures involved in replication since it is among the smallest and simplest of the RNA viruses. More importantly, TCV is associated with very small subviral RNAs that can be used in replication studies. Protoplast and in vitro systems have been developed to study replication of TCV genomic and subviral RNAs and several regions involved in replication of the subviral RNA satC have been identified. The TCV RdRp undergoes a process called abortive initiation when using TCV as template, which produces short RNA sequences able to function as primers for repair of deletions of satC ends. Preliminary results suggest that promoters at the ends of TCV and satC strands may not be functioning as units independent of upstream sequence as previously thought, but rather may be involved in specific interactions with interior sequences. To address this hypothesis, the sequence/structural requirements for abortive initiation from the TCV hairpin promoter will be determined by analyzing the importance of the 3' single-stranded tail and an upstream sequence element in abortive initiation by the TCV RdRp. This objective should permit the delineation of an element required for abortive initiation on the TCV genome. Mutations that are able to restore replication of TCV with the promoter from satC and that are outside the promoter sequence will be mapped by replacing segments of the mutant TCV into TCV with the promoter of satC and assaying for infectivity. This objective should provide another means for elucidating internal cis-acting sequences that interact with the 3' terminal promoter of TCV. In addition, the importance of an interior sequence in satC required for transcription in the absence of the 3' end bases will be investigated. Successful completion of these experiments should provide significant advances in our understanding of the function of RdRp's and their ability to recognize a wide variety of linear and structural elements as promoters for complementary strand synthesis.