Noncoding Region Between the Env and Src Genes of Rous Sarcoma Virus Influences Splicing Efficiency at the Src Gene 3' Splice Site

Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed.