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 the env and src genes and downstream of src in 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 and env mRNA 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.
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