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Journal of Virology, March 2004, p. 2517-2529, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2517-2529.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference Directed against Human Transcription Elongation Factor P-TEFb (CDK9/CyclinT1)

Ya-Lin Chiu,1 Hong Cao,1 Jean-Marc Jacque,2 Mario Stevenson,2 and Tariq M. Rana1*

Department of Biochemistry and Molecular Pharmacology,1 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 016052

Received 29 July 2003/ Accepted 7 November 2003

The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605. Phone: (508) 856-6216. Fax: (508) 856-8015. E-mail: tariq.rana{at}umassmed.edu.

Journal of Virology, March 2004, p. 2517-2529, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2517-2529.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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