Abstract
Equine infectious anemia virus (EIAV) activates transcription via a Tat protein, a TAR element, and the equine elongation factor positive transcription elongation factor b (P-TEFb). In human cells, EIAV Tat (eTat) can inhibit the ability of human immunodeficiency virus type 1 (HIV-1) Tat (hTat) to activate transcription from the HIV-1 long terminal repeat, demonstrating that EIAV Tat can interact nonproductively with human P-TEFb. To study the mechanism of EIAV Tat and HIV-1 Tat activation, we developed an in vitro elongation assay that recapitulates EIAV Tat-mediated inhibition of HIV-1 Tat trans-activation. We found that eTat specifically inhibits activation of elongation by HIV-1 Tat while having no effect on basal transcription elongation. The competitive inhibition of hTat activation was reversed by an activity present in HeLa cell nuclear extracts, most likely a form of P-TEFb. Recombinant P-TEFb (cyclin T1 and CDK9) overcame the inhibition of transcription by eTat but in a nonspecific manner. EIAV Tat affinity chromatography was used to purify the activity present in nuclear extract that was capable of reversing eTat inhibition. We characterized the protein components of this activity, which include cyclin T1, CDK9, Tat-SF1, and at least three unidentified proteins. These data suggest that additional factors are involved in the mechanism of Tat activation. (C) 2000 Academic Press.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 356-366 |
| Number of pages | 11 |
| Journal | Virology |
| Volume | 274 |
| Issue number | 2 |
| DOIs | |
| State | Published - Sep 1 2000 |
| Externally published | Yes |
Bibliographical note
Funding Information:We thank C. Hernández-Munain, P. Bieniasz, P. Rajaii, and H. Bogerd for their help during the course of this work. We also thank E. Wagner, P. Bohjanen, W. Maury, and R. Carstens for critical review of the manuscript; B. Cullen for plasmids and for communicating data before publication; and K. Jones for α-cyclinT1 antibodies. This research was supported by a grant from the National Institutes of Health to M.A.G.-B. C.S. was supported by the NIAID Research Training Program in AIDS to the Division of Infectious Diseases at Duke University Medical Center. The authors also acknowledge the Keck Foundation for support to the Levine Science Research Center.
