The role of Rev-SR protein interactions in the regulation of equine infectious anemia virus replication

Park, Gregory
Major Professor
Susan L. Carpenter
Committee Member
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Veterinary Microbiology and Preventive Medicine

Equine infectious anemia virus (EIAV) is a member of the lentivirus subfamily of retroviruses that produces a variable clinical disease course characterized as acute, chronic, and inapparent. The clinical signs can vary according to the stage of disease, and generally correlate with levels of virus replication. As with other retroviruses, EIAV utilizes both RNA and proteins to produce alternatively spliced transcripts required for virus replication. EIAV encodes a protein called Rev, which functions by binding unspliced and singly spliced viral mRNAs in the nucleus at a sequence called the Rev responsive element (RRE) and exporting them into the cytoplasm. Rev is absolutely required for virus replication, and factors that inhibit Rev function would be expected to inhibit virus replication. EIAV Rev is encoded in exons 3 and 4 of a bicistronic, four-exon mRNA, which also encodes the protein Tat in exons 1 and 2. The presence of Rev results in the expression of an alternatively spliced viral mRNA that differs from the four-exon mRNA by lacking exon 3. Exon 3 contains cis-acting sequences that function as both an exon splicing enhancer (ESE) and a RRE. ESEs bind cellular SR proteins to assist in the recognition and inclusion of exons. Therefore, the EIAV ESE/RRE sequences bind both SR proteins and Rev. The goal of this research is to characterize the interactions between EIAV and cellular SR proteins that modulate virus replication. I first show that Rev-mediated alternative splicing of exon 3 is not a mechanism to up-regulate Tat activity. I demonstrate that SF2/ASF inhibits Rev-mediated nuclear export activity and EIAV replication in vitro. I show that the RNA binding domain of SF2/ASF is necessary and sufficient for the inhibition of Rev nuclear export activity and EIAV replication. Further, the inhibition of both Rev activity and virus replication correlated with the SR protein RNA binding specificity. These results suggest that SR proteins and Rev compete for binding viral RNAs at the ESE/RRE. Therefore, factors that modulate intracellular concentrations of SR proteins may play a role in regulating Rev nuclear export activity and EIAV replication.