Characterization of alternative reading frame selection by a viral internal ribosome entry site

Dicistroviruses possess a positive-sense, monopartite single-strand RNA genome that encodes two open reading frames containing the nonstructural and structural polyproteins (ORF1 and ORF2) separated by the intergenic region (IGR) internal ribosome entry site (IRES). Translation of each ORF is directed by distinct IRESs, a 5’ untranslated region (UTR) and an IGR IRES. Previous bioinformatic studies have shown that a subset of dicistroviruses contain an overlapping gene in the +1 translational reading frame within the structural polyprotein gene near the IGR IRES region. We hypothesize that the IGR IRES directs translation of two overlapping ORFs, a novel +1 frame ORFx and the 0 frame ORF which encodes the viral structural polyprotein. In this thesis, using Israeli acute paralysis virus (IAPV) as a model, the existence and start site of ORFx were identified using mutagenesis and Mass Spectrometry analyses. In addition, the structural elements within the IAPV IGR IRES that determine alternative reading frame translation initiation were explored. Lastly, the localization of overexpressed tagged-ORFx in Drosophila S2 cells was examined to gain insights of its function. Summarizing, we have discovered a novel mechanism that increases the coding capacity of a virus through an IGR IRES. These studies of IAPV IGR-IRES will further our understanding of IRESs mediated translation initiation and reading frame decoding.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat