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J Virol, April 1998, p. 2615-2629, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Modulation of Sp1 Phosphorylation by Human Immunodeficiency Virus Type 1 Tat

Rene F. Chun, O. J. Semmes, Christine Neuveut, and Kuan-Teh Jeang*

Molecular Virology Section, Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0460

Received 8 August 1997/Accepted 19 December 1997

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67:6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.

* Corresponding author. Mailing address: Molecular Virology Section, Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Diseases, Building 4, Room 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Phone: (301) 496-6680. Fax: (301) 402-0226. E-mail: kjeang{at}atlas.niaid.nih.gov.




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