Binding of Nuclear Proteins to HTLV-II cis-Acting Repressive Sequence (CRS) RNA Correlates with CRS Function

Alexander C. Black, Cristina T. Ruland, Jie Luo, Andreas Bakker, John K. Fraser, Joseph D. Rosenblatt

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9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number71159
Pages (from-to)29-41
Number of pages13
JournalVirology
Volume200
Issue number1
DOIs
StatePublished - Apr 1994
Externally publishedYes

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ASJC Scopus subject areas

  • Virology

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