EQUINE INFECTIOUS-ANEMIA VIRUS TRANSREGULATORY PROTEIN REV CONTROLS VIRAL MESSENGER-RNA STABILITY, ACCUMULATION, AND ALTERNATIVE SPLICING

The cis and trans-acting components of the Rev regulatory pathway empl oyed by equine infectious anemia virus (EIAV) to regulate and coordina te viral gene expression were examined in complementation experiments. Viral protein expression and mRNA expression were compared in cells t ransiently transfected with wild-type or mutant proviruses in combinat ion with Rev expression plasmids. Mutation of the predicted rev gene a bolished Gag protein synthesis, and this defect was complemented, in t rans, by Rev. Analysis of viral mRNAs from transfected cells confirmed that EIAV expresses five major mRNAs: the full length and singly spli ced mRNAs contain introns and encode viral structural proteins while t he three fully spliced mRNAs, encoding nonstructural genes, are genera ted by alternative splicing. Compared to cells transfected with the wi ld-type provirus, the intron-containing mRNAs produced from the rev-mi nus mutant were present at reduced levels in the nuclear RNA fraction and were not detected in the cytoplasm. This pattern of viral mRNA syn thesis was restored to the wild-type pattern by providing Rev in trans . In contrast to the intron containing mRNAs, cytoplasmic accumulation of the multiply spliced class of mRNAs was independent of Rev. Closer examination of the multiply spliced class of viral mRNAs by reverse t ranscriptase-PCR analysis revealed a Rev-dependent alternative splicin g phenomenon. In the absence of Rev, proviruses expressed a four-exon mRNA at high levels; the addition of Rev caused both a decrease in the levels of the four-exon mRNA and the appearance of a related mRNA lac king exon 3. The cis-acting RNA elements that mediate Rev responsivene ss were studied with deleted proviruses, which revealed that EIAV cont ains at least two elements located near the ends of the envelope gene. Unlike the Rev-responsive elements in other retroviruses, the cis-act ing regions of EIAV do not appear to form complex secondary structures .