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

Research output: Contribution to journalArticle

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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
Pages (from-to)29-41
Number of pages13
JournalVirology
Volume200
Issue number1
DOIs
StatePublished - Jun 3 1994
Externally publishedYes

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Human T-lymphotropic virus 2
Human T-lymphotropic virus 1
Terminal Repeat Sequences
Nuclear Proteins
rex Gene Products
Carrier Proteins
RNA
Electrophoretic Mobility Shift Assay
Proteins
Gene Expression
Virology
Gels

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Binding of nuclear proteins to HTLV-II cis-acting repressive sequence (CRS) RNA correlates with CRS function. / Black, Alexander C.; Ruland, Cristina T.; Luo, Jie; Bakker, Andreas; Fraser, John K.; Rosenblatt, Joseph D.

In: Virology, Vol. 200, No. 1, 03.06.1994, p. 29-41.

Research output: Contribution to journalArticle

Black, Alexander C. ; Ruland, Cristina T. ; Luo, Jie ; Bakker, Andreas ; Fraser, John K. ; Rosenblatt, Joseph D. / Binding of nuclear proteins to HTLV-II cis-acting repressive sequence (CRS) RNA correlates with CRS function. In: Virology. 1994 ; Vol. 200, No. 1. pp. 29-41.
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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.",
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AB - 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.

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