Short Communication Dominant negative mutant cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat Julie K. Jadlowsky, 1 Masanori Nojima, 1 Takashi Okamoto 2 and Koh Fujinaga 1 Correspondence Koh Fujinaga kxf32@cwru.edu 1 Division of Infectious Diseases, Department of Medicine, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4984, USA 2 Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Received 8 April 2008 Accepted 2 July 2008 Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P- TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin- dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies. The transcription of the human immunodeficiency virus type 1 (HIV) is a highly regulated process in which several host cellular co-factors and the viral transactivator protein, Tat, are involved (Karn, 1999; Taube et al., 1999). Tat stimulates the elongation of transcription with the aid of the positive transcription elongation factor b (P-TEFb). The active form of P-TEFb is a heterodimer comprising cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9) (Peterlin & Price, 2006). Tat and CycT1 bind to the transactivation response element (TAR), an RNA stem– loop structure located at the 59 end (+1 to +59) of all HIV transcripts (Bieniasz et al., 1998; Fujinaga et al., 1999; Garber et al., 1998b). This interaction results in the recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat (Kim et al., 2002). Among 726 amino acids, the first 272 amino acids of CycT1 that form the cyclin box repeats are sufficient for HIV transcription, since these residues are responsible for the interactions with Tat, TAR and Cdk9 (Bieniasz et al., 1998, 1999; Fujinaga et al., 1999; Garber et al., 1998a, b; Ivanov et al., 1999). Since P-TEFb is the essential host cellular co-factor for Tat, it serves as a potential target for anti-HIV therapeutics. Several approaches have been taken to block HIV transcription by targeting P-TEFb (Richter & Palu, 2006), which include the use of small compounds or mutant proteins that inhibit Cdk9 kinase activity (Chao et al., 2000; Fujinaga et al., 2002; Heredia et al., 2005; Mancebo et al., 1997) or disrupt the interaction between Tat, TAR and CycT1 (Hwang et al., 2003; Lind et al., 2002; Mischiati et al., 2001; Okamoto et al., 2000), using intrabodies against CycT1 (Bai et al., 2003), or oligomerization chain reaction to inactivate Cdk9 (Napolitano et al., 2003). It is important to note, however, that since P-TEFb is involved in the transcription of many cellular genes (Chao & Price, 2001), it is critical to exclusively block HIV-specific pathway(s) in order to develop safe and effective anti- HIV therapies. Construction of mutant cyclin proteins that show dom- inant negative effects against endogenous cyclin–Cdk complexes has been quite challenging. This is presumably because the precise mechanism by which these cyclins activate the kinase activity of their corresponding Cdks is not fully understood. In order to clarify the structure– function relationship of CycT, which is critical for development of effective dominant negative CycT1 mutants, we have previously determined the crystal structure of the cyclin box region of CycT1 (Anand et al., 2007). The resultant three-dimensional structure indicated that CycT1 shares structural similarities with other cyclins Supplementary figures are available with the online version of this paper. Journal of General Virology (2008), 89, 2783–2787 DOI 10.1099/vir.0.2008/002857-0 2008/002857 G 2008 SGM Printed in Great Britain 2783