Repeated B motifs in the human immunodeficiency virus type I long terminal repeat enhancer region do not exhibit cooperative factor binding

R. B. Gaynor, M. D. Kuwabara, F. K. Wu, J. A. Garcia, D. Harrich, M. Briskin, R. Wall, D. S. Sigman

Research output: Contribution to journalArticle

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Abstract

The enhancer element of the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) contains two copies of nearly identical sequences AGGGACTTTCC (3G sequence) and GGGGACTTTCC (4G sequence) that are important in transcriptional regulation. A single copy of the 4G sequence is found in the NF-κB site of the immunoglobulin κ-chain enhancer. Only the 4G motif in the HIV enhancer is bound by cellular proteins in extracts prepared from unstimulated HeLa cells, whereas the 3G and 4G motifs are bound by factors in extracts prepared from HeLa cells treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA)] and lymphoid cells. To determine if this change in binding to the HIV enhancer was due to phosphorylation of a cellular protein, partially purified PMA-treated HeLa nuclear extracts were digested with calf intestinal phosphatase. Phosphatase digestion of nuclear extracts from PMA-treated HeLa cells markedly decreased factor binding to the HIV enhancer. Accordingly, phosphorylation of the DNA binding protein itself, or an inhibitor protein present in the partially purified extract, must mediate binding to the recognition sequence. Binding studies confirmed that each of the enhancer sequences was capable of binding factors independent of the activity of the other site and that the HIV enhancer was occupied by only one factor at any one time. Chloramphenicol acetyltransferase assays using mutants in either one or both HIV enhancer repeats revealed that each site was capable of functioning as a tat-inducible enhancer element in PMA-treated HeLa cells. These results suggest that the 3G and 4G motifs in the HIV enhancer function independently and that duplication in the HIV enhancer augments activity by a mechanism distinct from cooperative binding of NF-κB.

Original languageEnglish (US)
Pages (from-to)9406-9410
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number24
StatePublished - 1988

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HIV Enhancer
HIV Long Terminal Repeat
HeLa Cells
Acetates
Phosphoric Monoester Hydrolases
Immunoglobulin Subunits
Phosphorylation
Proteins
Chloramphenicol O-Acetyltransferase
DNA-Binding Proteins
Phorbol Esters
Digestion
Lymphocytes

ASJC Scopus subject areas

  • General
  • Genetics

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Repeated B motifs in the human immunodeficiency virus type I long terminal repeat enhancer region do not exhibit cooperative factor binding. / Gaynor, R. B.; Kuwabara, M. D.; Wu, F. K.; Garcia, J. A.; Harrich, D.; Briskin, M.; Wall, R.; Sigman, D. S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 24, 1988, p. 9406-9410.

Research output: Contribution to journalArticle

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