Research Expertise and Interest

biochemistry of HIV gene expression, transcriptional elongation, Tat activation, stage of transcriptional elongation, HIV replication, anti-HIV therapy

Research Description

We are interested in the molecular mechanisms controlling HIV gene expression. Specifically, we investigate how Tat, an essential regulatory protein encoded by the HIV virus, and its host cellular cofactors regulate HIV gene expression at the stage of transcriptional elongation. The biochemical function of Tat and its cofactors is crucial for HIV replication and could potentially be a target for anti-HIV therapy. Study of Tat activation of HIV transcriptional elongation will also reveal general mechanisms of gene regulation at the stage of elongation, an important control step still poorly understood.

Current Projects

The human positive transcription elongation factor P-TEFb, consisting of a Cdk9-cyclin T1 heterodimer, functions as a general as well as a Tat-specific transcription factor. P-TEFb activates transcription by phosphorylating RNA polymerase (Pol) II, leading to the formation of processive elongation complexes. As a Tat co-factor, P-TEFb stimulates HIV transcription by interacting with Tat and the TAR RNA structure located at the 5' end of the nascent viral transcript.

A major goal of our research is to elucidate the mechanisms controlling the P-TEFb activity in the cell. Toward this goal, we have recently identified 7SK, an abundant and evolutionarily conserved small nuclear RNA, as a specific P-TEFb-associated factor. 7SK inhibits general and Tat-specific transcriptional activities of P-TEFb by inhibiting the kinase activity of Cdk9 and preventing recruitment of P-TEFb to the HIV promoter. Under certain stress conditions, the 7SK:P-TEFb interaction is disrupted, resulting in a significant increase in HIV transcription and Pol II phosphorylation. Thus, the 7SK:P-TEFb interaction may function as a sensor to control stress-induced general and HIV-specific transcription. We are performing structure-function analyses of the 7SK:P-TEFb interaction to study the mechanism of 7SK inhibition of the Cdk9 kinase. We are also investigating the stress-induced signaling pathway controlling the 7SK:P-TEFb interaction and the possible involvement of this pathway in HIV replication and the escape from viral latency.

Another human elongation factor important for general and HIV-specific transcription is Tat-SF1. An important objective of our research is to elucidate the mechanism by which Tat-SF1 cooperates with P-TEFb to stimulate transcription. Using HIV proviral DNA as a model template, we have recently identified a role of Tat-SF1 and its associated factors in coupling transcriptional elongation and pre-mRNA splicing. Tat-SF1 interacts with spliceosomal U snRNPs to form a multi-subunit complex, which is recruited by P-TEFb to the elongating Pol II to activate both transcription and splicing. Supporting the idea that the recruitment of U snRNPs near the elongating polymerase is important for transcription, inclusion of splicing signals in the nascent transcript further stimulates polymerase elongation. Presently, we are investigating the mechanism by which the Tat-SF1-associated splicing factors stimulate transcription.

In addition to its ability to stimulate HIV transcription, Tat is also known to induce apoptosis of mainly CD4+ T cells, which may contribute in part to the progressive depletion of these cells and consequently the loss of immune competence during HIV infection. Another ongoing project in the lab seeks to understand the mechanism of Tat-induced apoptosis. We have recently discovered a specific interaction of Tat with the ab-tubulin dimer and the polymerized microtubules. This interaction alters microtubule dynamics, leading to the activation of a mitochondria-dependent apoptotic pathway that is facilitated by the proapoptotic Bcl-2 family member Bim. We are employing a variety of biochemical and cell biology techniques to further delineate the apoptotic pathway induced by Tat-perturbation of microtubule dynamics.