Rous sarcoma virus (RSV) contains two ∼135-nt imperfect direct repeats composed of smaller repeats, dr1 (∼100 nt) and dr2 (∼36 nt), that are between theenvandsrcgenes and downstream ofsrcin the 3′ untranslated region, respectively. It has previously been shown that a Prague A RSV mutant in which both dr1 sequences are deleted is defective at several points in the virus life cycle, including unspliced RNA andenvmRNA stability, unspliced RNA transport, and virus particle assembly. A defect in unspliced RNA transport occurs because a cytoplasmic transport element is present within the dr1. We have suggested that the defect of particle production may arise from the failure of the unspliced RNA to be targeted to sites in the cytoplasm where its translation is favorable for Gag protein assembly. In this report, we have further investigated the function of the direct repeats by comparing virus mutants containing either a single upstream or downstream dr1 sequence. Both mutants were delayed in replication compared to the wild-type; the mutant with a single upstream dr1 (ΔDDR) is significantly more defective than the mutant with a single downstream dr1 (ΔUDR). While both mutants appear capable of efficiently transporting unspliced RNA to the cytoplasm, the ΔDDR mutant with only the upstream dr1 is defective in its ability to support Gag assembly and particle release. The replication defect cannot be repaired by placing the upstream dr1 at the location of the downstream dr1 in the 3′ untranslated region. A single point mutation in the upstream dr1 (U to C) restored replication and particle production to near normal levels. The results suggest that unspliced RNA transport and Gag assembly functions may be mediated by different elements within the dr1 and that the Prague A upstream dr1 is defective in the latter but not the former function.

Two approximately 135-nucleotide (nt) direct repeats flank the Rous sarcoma virus (RSV) oncogene src and are composed of two smaller repeats, dr1 (approximately 100 nt) and dr2 (approximately 36 nt). These sequences have been reported to contain cis-acting signals necessary for RNA packaging and elements that allow cytoplasmic accumulation of unspliced RNA (cytoplasmic transport elements). In this report, we show that avian fibroblasts infected with the Prague A strain of RSV with precise deletions of both dr1 elements express src and are transformed by this mutant virus but production of virus particles is very low and virus spread throughout the culture requires several weeks. We show that the replication defect is due to complex effects on viral RNA transport, viral RNA half-life, and virus particle assembly. The dr1 elements may contain binding sites for a permissive cell-specific factor(s) that facilitates efficient nuclear-cytoplasmic transport, RNA stability, and cytoplasmic utilization of unspliced viral RNA. The implications of these results for understanding the defects of nonpermissive virus infections in mammalian cells are discussed.

All retroviruses require both unspliced and spliced RNA for a productive infection. One mechanism by which Rous sarcoma virus achieves incomplete splicing involves suboptimal env and src 3' splice sites. We have previously shown that mutagenesis of the nonconsensus src polypyrimidine tract to a 14-nucleotide uninterrupted polypyrimidine tract results in an oversplicing phenotype and a concomitant defective replication in permissive chicken embryo fibroblasts. In this report, we show that splicing at the src 3' splice site (3'ss) is further negatively regulated by the suppressor of src splicing cis element which is located approximately 100 nucleotides upstream of the src 3'ss. The increase in splicing at the src 3'ss results in a corresponding increase in splicing at a cryptic 5'ss within the env gene. Two classes of replication-competent revertants of the src oversplicing mutant (pSAP1) were produced after infection, and these mutants were characterized by molecular cloning and sequence analysis. Class I revertants are transformation-defective revertants in which the src 3'ss and the src gene are deleted by homologous recombination at several different sites within the imperfect direct repeat sequences that flank the src gene. Cells infected with these transformation-defective revertants produce lower levels of virus particles than cells infected with the wild-type virus. Class II revertants bear small deletions in the region containing the branchpoint sequence or polypyrimidine tract of the src 3'ss. Insertion of these mutated sequences into pSAP1 restored inefficient splicing at the src 3'ss and efficient replication in chicken embryo fibroblasts. All of these mutations caused reduced splicing at the src 3'ss when they were tested in an in vitro splicing system. These results indicate that maintenance of a weak src 3'ss is necessary for efficient Rous sarcoma virus replication.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.