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The human immunodeficiency virus (HIV) requires cellular proteins for its efficient replication in infected cells. Therefore, the identification and characterization of these co-factors would provide important information in order to understand the HIV life cycle and to develop efficient anti-viral therapies. In our laboratory, we focus mainly on the mechanism of HIV transcription and the characterization of two cellular protein complexes that positively and negatively regulate the elongation step of transcription.
1. P-TEFb (Positive transcription elongation factor b): P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclins T1, T2 or K, is the essential co-factor for HIV transcription. The cyclin T1 (CycT1) subunit of P-TEFb binds to the viral transactivator (Tat) protein and to the transactivation response (TAR) element, an RNA stem-loop structure located at the 5' of viral transcripts. This interaction results in the hyperphosphorylation of the C-terminal Domain (CTD) of RNA polymerase II (RNAPII) by Cdk9, which is necessary for the high processivity of RNAPII. We define this interaction in detail in order to construct effective dominant negative P-TEFb mutants that can disrupt this interaction and inhibit HIV transcription. In addition, since P-TEFb is important for the expression of many of cellular genes (such as oncogenes), we are studying the general role of this complex. We will identify the cellular genes that are regulated by P-TEFb. Also, other cellular proteins that interact with P-TEFb will be identified and characterized.
2. NELF (Negative elongation factor): In the absence of Tat, HIV transcription is kept at a very low level, which represents one possible mechanism for HIV latency in infected cells. We have previously demonstrated that the negative elongation factor (NELF) can be recruited to the HIV promoter in the absence of Tat by direct interaction between its RD subunit and TAR RNA. NELF induces a premature arrest of RNAPII. This negative effect can be alleviated in the presence of Tat and P-TEFb by phosphorylation of the RD subunit by Cdk9, which explains the mechanism of the transition from the abortive to the productive transcription of HIV. We will further study this interaction in order to construct mutant NELF complexes that cannot be removed by Tat/P-TEFb and therefore inhibit HIV transcription.
3. Several other HIV-related and cancer-related projects are being undertaken in our laboratory. These include: screening of HIV inhibitors, anti-HIV gene therapies, identification of other cellular proteins required for HIV replication, structural studies of the Tat/TAR/P-TEFb complex, and control of oncogene expression in tumor cells, among others.
Selected Publications
Wittman B.M., Fujinaga K., Deng H., Ogba N., and Montano M.M. The breast cell growth inhibitor, estrogen down-regulated gene 1 (EDG1), modulates a novel functional interaction between estrogen receptor alpha and transcription elongation factor cyclin T1. Oncogene (2005) 1-13. [PubMed]
Fujinaga K., Irwin D., Huang, Y., Taube R., Kurosu T., and Peterlin B. M. Dynamics of HIV transcription; P-TEFb phosphorylates RD and dissociates negative factors from TAR. Mol. Cell. Biol. (2004) 24:787-795. [PubMed]
Fujinaga K., Irwin D., Taube R., Zhang, F., Geyer M., and Peterlin B. M. A minimal Chimera between human cyclin T1 and Tat binds TAR and activates HIVtranscription in murine cells. J. Virol.(2002) 76: 12934-12939. [PubMed]
Fujinaga, K., Irwin, D., Geyer, M., and Peterlin, B. M. Optimized chimeras between kinase innactive, mutant Cdk9 and truncated Cyclin T1 proteinsinhibit efficiently Tat transacrtivayion and HIV replication. J. Virol. (2002) 76:10873-10881. [PubMed]
Taube R., Lin X., Irwin D., Fujinaga K., and Peterlin B. M. Interaction between P-TEFb and the C-terminal domain of RNA Polymerase II Activates transcriptional elongation from sites upstream or downstream of target genes. Mol. Cell. Biol. (2002) 22: 321-331. [PubMed]
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