Molecular Analysis of Translational Readthrough in Tobacco necrosis virus-D

Plus-sense single-stranded RNA plant viruses are a large group of agriculturally and economically important plant pathogens. These viruses encode their own RNA-dependent RNA polymerase (RdRp), whose translation from the viral genome is critical for a successful viral infection. Due to the limited size of their genomes, RNA plant viruses have developed several alternative methods of gene expression, including stop codon recoding. Recoding events, such as readthrough, allow for the translating ribosome to bypass a termination signal by incorporating an amino acid from a near-cognate tRNA. Several plant viruses, including those within the Tombusviridae family, utilize readthrough to produce their RdRp. Using a combination of in vitro and in vivo techniques, the importance of several RNA sequences and structures on readthrough efficiency were studied in the Betanecrovirus Tobacco necrosis virus strain D (TNV D). The overall goal of this dissertation was to identify the RNA sequences and structures that are involved in promoting efficient translational readthrough of the TNV-D RdRp. In this study, I show that readthrough production of the TNV-D RdRp requires multiple local RNA structures, as well as two long-range intra-genomic base-pairing interactions. A large RNA stem-loop structure (termed RTSL), located immediately downstream from the readthrough site, was found to be critical for readthrough, and was functional only when base-paired via a long-range RNA-RNA interaction with a sequence in the genomic 3-untranslated region (3UTR). RNA elements positioned just upstream and downstream from the RTSL were also investigated, and both Pre- and Post-RTSL RNA structures were found to be important for efficient readthrough. Within the 3UTR, SLII, and an RNA pseudoknot structure involving the 3-terminus of the viral genome were both determined to be required for readthrough, as well as a second long-range RNA-RNA interaction involving the 3UTR. Finally, SLX, also located in the 3UTR, was not required for readthrough, but was important for viral RNA accumulation. Collectively, these findings have helped to expand our understanding of the RNA elements involved in the mechanism of TNV-D translational readthrough. These results have also provided possible insight into other viruses that utilize type-III translational readthrough as a gene expression strategy.