Wilhelm bernhard workshop on the cell nucleus

REGULATION OF PROCESSIVE TRANSCRIPTION

Zhou M.¹, Deng L.¹, Kashanchi F.¹, Shatkin A.² and Kumar A.<sup>1

1</sup>Department of Biochemistry & Molecular Biology, George Wahington University, Washington, D.C. and ²Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA.
An understanding of the molecular mechanism of transcription elongation is important not only for basic biology but also for developing specific inhibitors of gene expression. Our studies are based on transactivation of HIV-1 promoter. Tat protein is known to induce transcription elongation of HIV-1 promoter by recruiting positive elongation factor, P-TEFb (composed of cyclin dependent kinase, CDK9 and cyclin T1) to the TAR RNA structure at the 5’end of nascent viral transcripts. We asked, (i) how does Tat modify general transcription factor P-TEFb, to specifically induce processive transcription from HIV-1 promoter, and (ii) would an inhibitor of the Tat/TAR dependent P-TEFb kinase selectively block viral replication? Earlier studies suggested that HIV Tat stimulates transcription elongation by binding cyclin T1 to the TAR RNA domains of nascent viral transcripts. Since the escape from paused transcripts, which appears to be an important check point for RNA Pol II genes, has been linked to P-TEFb mediated phosphorylation of RNAP II CTD repeats, we asked whether the Tat/TAR RNA interaction induced P-TEFb kinase. Main points of the results that will be discussed are as follows: (i) Tat/TAR induced P-TEFb kinase specifically phosphorylates RNAP II CTD serine 5; (ii)Tat/TAR dependent P-CTD serine 5 stimulates co-transcriptional capping of nascent viral transcripts; (iii) phosphorylation of the transcription elongation factors, SPT5 and Tat-SF1, is dependent upon the Tat modified P-TEFb kinase; (iv) the CDK inhibitor, Flavopiridol blocks phosphorylation of SPT5 and Tat-SF1 as well as P-CTD serine 5 at identical kinetics (IC₅₀=1nM); (v) Chromatin immunoprecipitation assays showed that the capped mRNA, along with SPT5, Tat-SF1 and P-CTD serine 5 associate with the promoter proximal region of HIV-1 gene and are highly sensitive to Flavopiridol; and (vi) at the low concentrations of Flavopiridol that attenuates HIV-1 replication, there is negligible change in the expression levels of host cell genes as seen in microarray assays. Overall, the results argue that the Tat modified transcription factors provide the basis for specific induction of viral gene expression. One prediction of the results, that viral replication would be uniquely dependent on the transcription elongation factors, SPT5 and Tat-SF1 was tested by RNAi mediated post-transcriptional gene silencing. Results show that ablation of either SPT5 or Tat-SF1 with synthetic siRNA complementary to SPT5 and Tat-SF1 mRNAs, severely attanuates HIV-1 replication. These studies show that HIV-1 encoded Tat protein modifies transcription factors to stimulate viral gene expression and that the step can be selectively inhibited by a CDK9 inhibitor to block viral replication.. This approach opens the possibility of developing novel inhibitors that are based on host cell enzymes and thus unlikely to engender resistant viral strains.