Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ding S. Shih

Abstract

Potato Virus X is the type member of the Potexvirus group. It is a flexuous rod containing a single-stranded, positive-sense RNA. Previous studies have shown that during infection Potato Virus X synthesizes subgenomic RNAs to express internal cistrons. These studies demonstrated that six subgenomic RNAs could be detected in total RNA extracts from PVX-infected tissue; however, only four internal ORFs are predicted within the genomic RNA. Furthermore, two particle size populations were detected, one containing genomic RNA and one containing the coat protein mRNA. In the present investigation experiments were designed to examine PVX-encoded subgenomic RNAs with regard to their total number, their coding properties, and possible encapsidation. Electron microscopic examination of crude extracts and partially purified virus preparations of PVX revealed that the virus particles exist in seven distinct size classes using a published virus isolation procedure developed for PVX. For analysis of the RNA species encapsidated in the smaller particles, virus purification and RNA extraction procedures were developed to allow reproducible isolation of the smaller RNA species. Using these procedures, seven RNA species (6.4 kb, 3.6 kb, 3.0 kb, 2.1 kb, 1.8 kb, 1.4 kb, and 0.9 kb) were detected from purified virus preparations by agarose gel electrophoresis. Northern blot analysis using two virus-specific probes containing either the 5$\sp\prime$ or 3$\sp\prime$ terminal sequences indicated that the smaller RNA species were 3$\sp\prime$ coterminal but shared no homology with the 5$\sp\prime$ end. PVX viral RNAs were selected from an oligo dT column indicating the presence of a poly A tail. Translation of PVX viral RNAs in wheat germ extracts and rabbit reticulocyte lysate resulted in the synthesis of at least four to five virus-specific proteins with relative masses corresponding to ORFs encoding polypeptides of sizes 165,588 (ORF 1), 24,547 (ORF 2), 12,589 (ORF 3), 7,943 (ORF 4), and 25,080 (ORF 5). Western blot analysis of proteins extracted from infected tissue using antisera prepared to virus-specific peptides indicated that at least five viral proteins are produced in vivo.

Pages

156

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