Identification of critical elements for dicistrovirus IGR IRES mediated translation

All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an InterGenic Region Internal Ribosome Entry Site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that is comprised of a ribosome binding domain (PKII and PKIII) and a tRNA-like anticodon domain (PKI) connected via a short, 1-3 nucleotide hinge. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In chapter 2, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. The dicistrovirus, Cricket paralysis virus (CrPV), has evolved a novel recoding mechanism whereby ribosomes recruited to the viral internal ribosome entry site (IRES) undergo a ribosome bypass event to either start translation or continue translation 37 nucleotides downstream to direct +1 frame translation of an ORF called ORFx. Although ribosome bypassing has been described in bacteriophage T4 gene 60 and in mitochondria of yeast, to the best of our knowledge, the ribosome bypassing mediated by the CrPV IRES is the first to be reported in higher order eukaryotes. In chapter 3, we employ a mutational analysis to identify genomic elements contributing to IRES mediated ribosome bypassing.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat