Date of Award

1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ding S. Shih

Abstract

The 3.5-kb RNA of equine infectious anemia virus (EIAV) is shown here to encode the S1, S2 and envelope proteins in vitro. S1 is expressed from this RNA despite the lack of an AUG codon. Deletion mutants of a cDNA derived from the 3.5-kb RNA were used to determine that sequences within the first 14 codons of ORF S1 are required for S1 expression in vitro and trans-activation in vivo. Amino-terminal sequence analysis of the in vitro synthesized S1 protein is consistent with initiation at a CUG codon. The translation properties of the 3.5-kb RNA have been examined in wheat germ extracts. The cap-dependence of the three cistrons was examined by addition of m$\sp7$GpppG cap analog to the wheat germ extract translation mixtures. The results indicated a near proportionate reduction in the expression of each of the cistrons suggesting that their translation is dependent on cap binding followed by ribosome scanning. The relative efficiencies of expression of the three cistrons were examined in translation reactions where the concentrations of magnesium (Mg$\sp{++}$) and potassium (K$\sp+$) ions were varied. Increasing the level of Mg$\sp{++}$ appeared to favor translation of the S1 protein, whereas increasing the K$\sp+$ concentration resulted in significantly reduced translation of S1 with concomitant enhanced translation from the downstream cistrons. The in vitro synthesized S2 protein was immunoprecipitated with sera obtained from EIAV-infected horses demonstrating that this protein is expressed in vivo. A 20-kd protein produced in vitro and in vivo from a Rev (S3) cDNA was immunoprecipitated with a monoclonal antibody directed against an epitope of the gp90 envelope protein. This finding indicates that env gene sequences provide a translational initiation codon for Rev protein expression. The Rev protein could be detected in EIAV-infected cells within 20 hours after infection and appeared to reach a maximal level of expression between 2 and 5 days post-infection.

Pages

210

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