THE UPSTREAM DIRECT REPEAT SEQUENCE OF PRAGUE-A ROUS-SARCOMA VIRUS ISDEFICIENT IN MEDIATING EFFICIENT GAG ASSEMBLY AND PARTICLE RELEASE

Rous sarcoma virus (RSV) contains two similar to 135-nt imperfect dire ct repeats composed of smaller repeats, dr1 (similar to 100 nt) and dr 2 (similar to 36 nt), that are between the env and src genes and downs tream of src in the 3' untranslated region, respectively. It has previ ously been shown that a Prague A RSV mutant in which both dr1 sequence s are deleted is defective at several points in the virus life cycle, including unspliced RNA and env mRNA stability, unspliced RNA transpor t, and virus particle assembly. A defect in unspliced RNA transport oc curs because a cytoplasmic transport element is present within the dr1 . We have suggested that the defect of particle production may arise f rom the failure of the unspliced RNA to be targeted to sites in the cy toplasm where its translation is favorable for Gag protein assembly. I n this report, we have further investigated the function of the direct repeats by comparing virus mutants containing either a single upstrea m or downstream dr1 sequence. Both mutants were delayed in replication compared to the wild-type; the mutant with a single upstream dr1 (Del ta DDR) is significantly more defective than the mutant with a single downstream dr1 (Delta UDR). While both mutants appear capable of effic iently transporting unspliced RNA to the cytoplasm, the Delta DDR muta nt with only the upstream dr1 is defective in its ability to support G ag assembly and particle release. The replication defect cannot be rep aired by placing the upstream dr1 at the location of the downstream dr 1 in the 3' untranslated region. A single point mutation in the upstre am dr1 (U to C) restored replication and particle production to near n ormal levels. The results suggest that unspliced RNA transport and Gag assembly functions may be mediated by different elements within the d r1 and that the Prague A upstream dr1 is defective in the latter but n ot the former function. (C) 1998 Academic Press.