Date Available

7-13-2014

Year of Publication

2014

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant Pathology

First Advisor

Dr. Said A. Ghabrial

Abstract

Double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two large open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), was previously proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In this study, I provided evidence to confirm that coupled termination-reinitiation (stop-restart) is indeed used. A dual-fluorescence method was established to define the RNA sequence determinants for RdRp translation. Stop-restart depends on a 32-nt stretch of RNA sequence immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The presence of similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop–restart strategy for RdRp translation. The close proximity of the secondary structure to the AUGA motif appears to be especially important for promoting translation of the downstream ORF. Normal strong preferences for AUG start codons and canonical sequence context for translation initiation of the downstream ORF appear somewhat relaxed. With dual-fluorescence system, reinitiation efficiency of the downstream ORF was determined to be ~3.9%. Pseudoknot swapping between the one in HvV190S and those predicted from other victoriviruses showed that reinitiation from the downstream ORF of HvV190S is quite tolerant to varying primary sequences of the various pseudoknots. Mutational analysis by introducing different combinations of nucleotide mutations into pseudoknot stems reproducibly confirmed the determinant role of pseudoknot on reinitiation using two different experimental systems. Together, these results provide the first example of coupled termination-reinitiation regulated by a simple pseudoknot stucture. These data expanded the understanding of coupled termination-reinitiation mechanism employed by RNA viruses and refined a new model for genus victorivirus, the largest genus in the family Totiviridae. The dual fluorescence system used in this study represented the first application of an efficient in vivo assay for recording low-frequency events in filamentous fungi.

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