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Journal of Virology, December 2002, p. 12934-12939, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12934-12939.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

Koh Fujinaga, Dan Irwin, Ran Taube, Fan Zhang, Matthias Geyer, and B. Matija Peterlin*

Departments of Medicine, Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0703

Received 13 May 2002/ Accepted 16 September 2002

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5' end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.

* Corresponding author. Mailing address: UCSF-Mt. Zion Cancer Center, Room N225, 2340 Sutter St., San Francisco, CA 94115. Phone: (415) 502-1902. Fax: (415) 502-1901. E-mail: MATIJA{at}ITSA.UCSF.EDU.

Journal of Virology, December 2002, p. 12934-12939, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12934-12939.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Schulte, A., Czudnochowski, N., Barboric, M., Schonichen, A., Blazek, D., Peterlin, B. M., Geyer, M. (2005). Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat. J. Biol. Chem. 280: 24968-24977 [Abstract] [Full Text]  
  • Fujinaga, K., Irwin, D., Huang, Y., Taube, R., Kurosu, T., Peterlin, B. M. (2004). Dynamics of Human Immunodeficiency Virus Transcription: P-TEFb Phosphorylates RD and Dissociates Negative Effectors from the Transactivation Response Element. Mol. Cell. Biol. 24: 787-795 [Abstract] [Full Text]  
  • Lin, X., Irwin, D., Kanazawa, S., Huang, L., Romeo, J., Yen, T. S. B., Peterlin, B. M. (2003). Transcriptional Profiles of Latent Human Immunodeficiency Virus in Infected Individuals: Effects of Tat on the Host and Reservoir. J. Virol. 77: 8227-8236 [Abstract] [Full Text]  


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