Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation

J. A. Garcia, D. Harrich, E. Soultanakis, F. Wu, R. Mitsuyasu, R. B. Gaynor

Research output: Contribution to journalArticle

166 Citations (Scopus)

Abstract

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression.

Original languageEnglish (US)
Pages (from-to)765-778
Number of pages14
JournalEMBO Journal
Volume8
Issue number3
StatePublished - 1989

Fingerprint

HIV Long Terminal Repeat
Viruses
HIV-1
Chemical activation
Mutagenesis
Mutation
Gene expression
DNA-Binding Proteins
Nucleic Acid Repetitive Sequences
Oligonucleotides
RNA-Binding Proteins
Assays
DNA
Carrier Proteins
Binding Sites
RNA
Gene Expression
Protein Binding
Base Pairing
Untranslated Regions

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Garcia, J. A., Harrich, D., Soultanakis, E., Wu, F., Mitsuyasu, R., & Gaynor, R. B. (1989). Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. EMBO Journal, 8(3), 765-778.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. / Garcia, J. A.; Harrich, D.; Soultanakis, E.; Wu, F.; Mitsuyasu, R.; Gaynor, R. B.

In: EMBO Journal, Vol. 8, No. 3, 1989, p. 765-778.

Research output: Contribution to journalArticle

Garcia, JA, Harrich, D, Soultanakis, E, Wu, F, Mitsuyasu, R & Gaynor, RB 1989, 'Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation', EMBO Journal, vol. 8, no. 3, pp. 765-778.
Garcia, J. A. ; Harrich, D. ; Soultanakis, E. ; Wu, F. ; Mitsuyasu, R. ; Gaynor, R. B. / Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. In: EMBO Journal. 1989 ; Vol. 8, No. 3. pp. 765-778.
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