Promoter-proximal pausing of RNA polymerase II (Pol II) on integrated HIV-1 proviral DNA has long been recognized as a major rate-limiting step in viral gene expression. To overcome this restriction, the HIV Tat protein recruits the human Super Elongation Complex (SEC) to paused Pol II through forming a multi-subunit complex on the TAR RNA, a hairpin structure located at the 5' end of all viral transcripts. SEC contains two powerful transcription elongation factors P-TEFb and ELL2 that act by different mechanisms but can synergize in their stimulation of Pol II elongation to generate full-length HIV transcripts. Notably, the identification of the SEC as a Tat cofactor was performed in the absence of TAR. It is unclear whether this RNA element simply serves as a platform to recruit Tat-SEC to the viral promoter, or as suggested by our preliminary data, may play additional regulatory roles by attracting other yet-to-be identified factors. To better understand the mechanism controlling TAR-specific and Tat- dependent HIV transcription, we propose to use a combination of in vivo and in vitro affinity-purification approaches to isolate and identify new components of the TAR RNPs and investigate their functional significance and mechanism of action in HIV gene expression. In addition to existing in the SEC, P-TEFb is also found in another catalytically active complex containing BRD4, a well-known BET bromodomain protein, epigenetic reader and major cancer therapeutic target. Although the BRD4 recruitment of P-TEFb is required for transcription of many cellular especially cancer-related genes, it is inhibitory to Tat-transactivation because BRD4 and Tat complete for binding to P-TEFb. Recently, we have identified REJ, a natural product derived from a traditional Chinese medicinal herb, which can antagonize BRD4 inhibition of Tat function through possibly activating a specific ubiquitin-proteasome pathway to selectively degrade BRD4. Based on these data, another major objective of this proposal is to investigate the mechanism by which REJ activates Tat-dependent HIV transcription and also to elucidate the proteolytic pathway that is induced by REJ to degrade BRD4. The identification of key components of this pathway especially the BRD4-specific E3 ubiquitin ligase will allow us to selectively control the cellular BRD4 level for potential therapeutic intervention in HIV/AIDS and cancer.
The Tat protein encoded by the HIV-1 virus activates transcription from the integrated proviral DNA by binding to the TAR RNA structure located at the 5' end of nascent viral transcripts. Although Tat is known to recruit a key human co-factor called the SEC to TAR, other unknown proteins may also exist on TAR to further modulate Tat activation of HIV transcription. On the other hand, Tat function can also be inhibited by a cellula protein called BRD4, and our preliminary data suggest that this inhibition can be antagonized by REJ, a novel product derived from a traditional Chinese medicinal herb. In this proposal, we plan to identify all the components of the TAR ribonucleoprotein complexes and investigate their functional significance in HIV gene expression. Furthermore, we will also investigate the mechanism by which REJ inhibits BRD4 to promote Tat-dependent HIV transcription. The proposed study may reveal new targets and methods for suppressing HIV replication.
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