Investigation of novel elements in dicistrovirus IGR IRES-mediated translation

Viruses have evolved a variety of mechanisms to subvert cellular processes, specifically at the point of translation, a highly regulated process that relies on recognition of the 5’-cap. One subversive mechanism that many RNA viruses use is the internal ribosome entry site (IRES), a RNA secondary structure that can bypass the necessity of the 5’-cap, translation factors, and even AUG start codon. The most streamlined IRESs are intergenic region (IGR) IRESs, derived from dicistroviruses, where it resides between two main viral open reading frames (ORFs). Interestingly, a subset of dicistroviruses have been identified to contain a +1-frame open reading frame (ORFx) that overlaps the open reading frame 2 (ORF2). Recent studies have indicated some possible mechanisms for +1 frame translation and the role of the resulting protein, but much of it remains elusive. In chapter 2, I evaluated dataset of dicistro-like viruses for predicted +1 open reading frame ORFx. Further, I examined the mechanism of the +1-frame ORFx translation from black queen cell virus (BQCV) IGR IRES, which has previously exemplified high ratios of translation. I determined that BQCV +1-frame translation is IGR IRES-dependent and driven by the downstream sequence. As well, I provide evidence that the BQCV IGR IRES-mediated +1-frame translation is dependent on translation of the 0-frame protein. I propose a model wherein translation begins in the 0-frame and frameshifts downstream into +1-frame. Recently, metagenomic screens of environmental samples have identified previously unknown dicistroviruses and dicistro-like viruses that contain an IGR IRES. Some of these genomes have uncommon structural and organizational characteristics. Specifically, some reside in coding regions of the first open reading frame (ORF1). In chapter 3, two novel predicted IGR IRESs, DS1 and DS2, are evaluated for functionality in vitro on their ability to bind to ribosomes and translation. Further, these are evaluated on how genome arrangement affects translation and translational regulation of the first and second open reading frames. Lastly, these IGR IRESs are analyzed for important structural motifs, providing insights on the similarity of these mechanisms to previously studied IGR IRESs.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat