Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Ronald C. Montelaro

Abstract

The transcriptional properties of equine infectious anemia virus were examined in two distinct equine cell lines in which the virus establishes either a persistent or a cytopathic infection. Northern hybridization analyses were performed to determine the number, sizes, and relative levels of the EIAV transcripts encoded during persistent or cytopathic infections. Three species of viral mRNA were detected in infected cells: an 8.2-kb full-length genomic mRNA, a 3.5-kb single-spliced mRNA, and a low abundance 1.5-kb mRNA, presumably formed by a double-splicing event of the full-length mRNA. Analysis of the levels of EIAV-specific RNA's present during persistent and cytopathic infections has revealed that quantitative differences characterize the transcriptional patterns of EIAV in these two infections. In persistently infected FEK cells the 8.2- and 3.5-kb mRNA's are the predominant viral transcripts and are detected in approximately equal concentrations, while the 1.5-kb mRNA is detected at very low levels. During the cytopathic infection of FDD cells, however, the 3.5-kb mRNA is the predominant viral transcript, comprising nearly 75% of the total viral mRNA, while the 8.2- and 1.5-kb mRNA's constitute the remaining 25% of viral transcripts. Moreover, the cytopathic infection is characterized by almost a thirty-fold higher level of viral transcripts than those detected during the persistent infection. The splicing patterns of the full-length EIAV mRNA during the cytopathic infection were determined by cDNA cloning and sequencing, Northern hybridization analyses using splice donor-specific oligonucleotide probes, and S1 nuclease mapping of RNA from virus-infected cells. The results have identified the splice donor and acceptor sites used to generate the spliced mRNA's of EIAV in infected cells. The expression of a putative regulatory protein of EIAV from a structural viral gene was investigated by analysis of in vitro and in vivo expression products. In vitro transcription and translation along with in vivo expression in transfected COS-7 cells were used to analyze the expression of the viral env gene. Based on the results of these studies, a potential mechanism for co-expression of two separate proteins from the env mRNA is proposed.

Pages

164

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