Genetic and biological variation in equine infectious anemia virus Rev correlates with variable stages of clinical disease in an experimentally infected pony.

Genetic and biological variation in the regulatory protein Rev of equine infectious anemia virus (EIAV) were examined throughout a clinically dynamic disease course of an experimentally infected pony. Following infection with the virulent EIAV(Wyo), the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (DPI), multiple recurrent fever episodes until 135 DPI, a prolonged subclinical period, and two late fever episodes. Viral RNA was isolated from the inoculum and sequential sera samples, and the rev exon 2/gp45 overlapping ORFs were amplified, cloned, and sequenced. Novel variants were found throughout infection, and genetic analyses indicated that both the Rev and gp45 ORFs were under selective pressure. The Rev variant predominant in the inoculum, R1, remained predominant during the early periods following infection (until 35 DPI); however, R1 was replaced by new predominant variants during the recurrent fever period (67-135 DPI). R1 reemerged as the predominant variant during the afebrile period, but a new predominant variant, R93, was associated with the late fever episodes. Rev variants predominant during recurrent febrile and late-febrile periods had significantly higher Rev-mediated nuclear export activity than the variants predominant during the acute and afebrile periods. Statistical correlation was found between Rev activity and different stages of clinical disease. Together, these results suggest that genetic and biological variation in rev may be a contributing factor in EIAV disease progression.

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Biological characterization of Rev variation in equine infectious anemia virus.

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.