Regulation of Viral Subgenomic mRNA-Mediated Gene Expression in Three RNA Plant Viruses

RNA virus genomes encode different viral proteins that are essential for establishing infections in their hosts. Expression of a subset of these proteins occurs from viral genome- derived subgenomic (sg) mRNAs that are transcribed during infections. The regulation of sg mRNA transcription and translation ensures that requisite amounts of each of these viral proteins are produced at specific stages of the infectious cycle. Higher-order RNA structures present within viral genomes and sg mRNAs are commonly used as molecular switches to achieve the necessary control. The goal of my dissertation was to investigate the structure and function of RNA elements involved in regulating sg mRNA-mediated gene expression in three plus-strand RNA plant viruses: carnation Italian ringspot virus (CIRV), pea enation mosaic virus 2 (PEMV2), and PEMV1. Structural and functional analyses, both in vitro and in cell infections, allowed for the delineation of distinct regulatory RNA elements in each virus. The RNA structures involved in activating sg mRNA transcription were investigated in CIRV and PEMV2. Activation of sg mRNA1 transcription in CIRV requires the formation of a large, complex, intragenomic higher-order RNA structure that assembles via a multistep folding pathway involving six long-distance RNA-RNA base-pairing interactions. In contrast, PEMV2 sg mRNA transcription involves a small RNA stem-loop that contains a self-complementary palindromic loop sequence. Transcription is activated by viral genome dimerization via an intergenomic kissing-loop interaction involving pairing of the palindromic sequences. In PEMV1, the RNA structure required to promote translation readthrough of a C-terminally extended capsid protein from its sg mRNA was investigated. A complex, non-contiguous RNA structure assembled by sequential formation of three long-distance RNA-RNA interactions was found to be required for this recoding event. Collectively, these results have uncovered several distinct regulatory RNA structures involved in controlling different aspects of sg mRNA-mediated viral gene expression and provide novel insights into RNA-based regulation in plus-strand RNA viruses.