Multiple regions in the Rous sarcoma virus src gene intron act in cis to affect the accumulation of unspliced RNA

Retrovirus replication requires the production of both spliced and unspliced viral RNA from a single RNA transcript. In contrast, cellular RNA precursors with introns almost completely spliced. The cis elements and virus-coded trans factors which determine the extent to which Rous sarcoma virus RNA is spliced to src mRNA were investigated by transfecting chicken embryo fibroblasts with cloned wild-type and mutant DNA followed by the analysis of viral RNA. Two cis-acting regions important in the negative control of splicing were detected. Cell cultures transfected with a clone deleted in 80% of the src intron (nucleotide 1149 to nucleotide 6574) demonstrated only a 2-fold reduction in the ratio of unspliced to spliced RNA relative to the wild-type clone, whereas cultures transfected with clones which were further deleted in the gag gene region (between nucleotide 630 and nucleotide 5258) demonstrated an approximate 20-fold reduction. Cell cultures which were transfected with clones deleted only between nucleotides 543 and 1806 demonstrated only a three- to fourfold reduction in the unspliced-to-spliced RNA ratio, suggesting that at least one other region of the src intron can partially compensate for the deletion of the gag region. Both regions appeared to act in cis on the distribution of unspliced and spliced RNA since the ratios were not altered by cotransfection with helper virus DNA.