BINDING OF NUCLEAR PROTEINS TO HTLV-II CIS-ACTING REPRESSIVE SEQUENCE(CRS) RNA CORRELATES WITH CRS FUNCTION

The shift from viral regulatory to structural gene expression in human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) is mediated b y Rex. We have previously shown that HTLV-II Rex acts through an eleme nt in R/U5 of the 5' long terminal repeat (LTR), the Rex-responsive el ement (RxRE), and that Rex protein binds to specific RNA sequences, th e Rex binding element (RBE), contained within the RxRE (Black et al., J. Virol. 65, 6645-6653, 1991b). Rex action through the RBE (nt 405-52 0) overcomes the inhibition of expression conferred by a contiguous LT R RNA regulatory element, which contains cis-acting repressive sequenc es (CRS; nt 520-630) that are not bound by Rex protein (Black et al., Virology, 181, 433-444, 1991a). We now show by electrophoretic mobilit y shift assay (EMSA) that cellular proteins in a HeLa nuclear extract bind specifically to RNA transcripts containing the HTLV-II CRS. Using ultraviolet (uv) crosslinking of gel-retarded bands, we identified a major protein species of approximately 60 kDa, p60CRS, that binds to C RS RNA and, with weaker affinity, to RBE RNA, In addition, a distinct 40-kDa protein, p40CRS, binds to U5 RNA (nt 645-750) downstream from t he CRS. Specific deletions within CRS RNA can reduce or abrogate bindi ng to this 60-kDa protein. EMSA and uv crosslinking assays also sugges t that both p60CRS and p40CRS interact with CRS RNA. CRS function in a 5' LTR-linked gene expression assay correlates with the ability of bo th p60CRS and p40CRS to interact with 5' LTR RNA in vitro, (C) 1994 Ac ademic Press, Inc.