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 by Rex. We have previously shown that HTLV-II Rex acts through an element in R/U5 of the 5' long terminal repeat (LTR), the Rex-responsive element (RxRE), and that Rex protein binds to specific RNA sequences, the Rex binding element (RBE), contained within the RxRE (Black et al., J. Virol. 65, 6645-6653, 1991b). Rex action through the RBE (nt 405-520) overcomes the inhibition of expression conferred by a contiguous LTR RNA regulatory element, which contains cis-acting repressive sequences (CRS; nt 520-630) that are not bound by Rex protein (Black et al., Virology, 181, 433-444, 1991a). We now show by electrophoretic mobility 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 CRS 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 the CRS. Specific deletions within CRS RNA can reduce or abrogate binding to this 60-kDa protein. EMSA and uv crosslinking assays also suggest that both p60CRS and p40CRS interact with CRS RNA. CRS function in a 5' LTR-linked gene expression assay correlates with the ability of both p60CRS and p40CRS to interact with 5' LTR RNA in vitro.
Black
A C
AC
Department of Medicine, UCLA School of Medicine 90024.
Ruland
C T
CT
Luo
J
J
Bakker
A
A
Fraser
J K
JK
Rosenblatt
J D
JD
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003432:Cross-Linking Reagents; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D015367:Human T-lymphotropic virus 2 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015500:Chloramphenicol O-Acetyltransferase