Doctoral Dissertations

Date of Award

5-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David L. Hacker

Committee Members

David Brian, Brad Reddick, Stuart Riggsby, Gary Stacey

Abstract

The cowpea strain of southern bean mosaic virus (SBMV-C) is a member of the sobemovirus group of positive-sense RNA viruses. A genome-length cDNA clone of SBMV-C was constructed and used as template for the in vitro synthesis of infectious SBMV-C RNA. Inoculation of Vigna unguiculata (cowpea) with the synthetic RNA resulted in a systemic infection of this host. The SBMV-C cDNA clone was used for the generation of mutations in three of the four SBMV-C open reading frames (ORFs). Synthetic transcripts generated from these mutant constructs were used to determine that the ORF1, ORF3, and coat protein genes are required for infectivity in cowpea but were not required for SBMV-C RNA synthesis in cowpea protoplasts. From these results it was concluded that these three proteins function in SBMV-C cell-to-cell movement. The requirement for the coat protein in cell-to-cell movement suggests that virus particles are transported locally. Studies were also carried out to determine the expression strategy of SBMV-C RNA. It was determined that the presence of cap analog decreased ORF1 and ORF2 expression in a cell-free extract, that the addition of AUG codons 5' to the ORF1 or ORF2 initiation codons decreased the translation of these genes in vitro and in vivo, and that deletions or insertions in ORF1, in general decreased ORF1 and ORF2 translation in vitro and in vivo. From these results it was concluded that ORF1 and ORF2 are expressed by a 5' end-dependent scanning mechanism rather than by internal ribosome entry.

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