Latent reservoirs of HIV-1 are the principal impediment to eradication of infection as they harbor transcriptionally silent proviruses that resume replication once therapy is disrupted. Methods are being developed to purge these reservoirs through reactivating latent HIV in the presence of HAART. However, the efficacy and specificity of the available latency activators are in need of major improvement, which can only be achieved through the identification and characterization of their relevant molecular target(s). This proposal explores the potential of targeting our recently identified Tat cofactors to activate latency. One widely studied Tat cofactor is P-TEFb, whose active form was recently shown to exist in a novel complex termed BFEC (bi-functional elongation complex) that also contains ELL2, AFF4, ENL and AF9. Within BFEC, AFF4 works as a scaffold to interconnect P-TEFb and ELL2, two well-known transcription elongation factors that act by distinct mechanisms. This synergistically activates elongation from many cellular and viral promoters, although the most prominent effect is on the HIV LTR. Importantly, Tat binds to BFEC to markedly enhance its formation and coordinate the actions of P-TEFb and ELL2 on the same polymerase enzyme to stimulate HIV transcription. ELL2 is normally a short-lived protein targeted by the proteasome. The Tat/AFF4-promoted BFEC formation stabilizes ELL2 in a process that requires ELL2's phosphorylation by probably P-TEFb. Finally, implicating a key role for BFEC in HIV latency activation, prostratin, HMBA and SAHA, the three most highly studied chemical activators of latency, are found to act like Tat to promote ELL2 expression and interaction with P-TEFb. These findings support the central hypothesis that the function and formation of BFEC can be promoted to reactivate latent HIV. To test this, we will examine whether active BFEC is both necessary and sufficient to reactivate HIV from latently infected T cell lines and primary CD4 cells. To generate degradation-resistant ELL2 highly potent for latency activation and control the upstream signaling pathway to further enhance this effect, we will identify the phosphorylation site(s) in ELL2 and the responsible kinase(s) that controls ELL2 stability and BFEC formation. Finally, to elucidate the proteolytic pathway that causes ELL2 degradation, we will test whether the ubiquitination of ELL2, which can be suppressed by Tat-induced phosphorylation, triggers ELL2 degradation by the proteasome. Major efforts will also be directed toward the identification of the ubiquitination enzymes specific for ELL2, which may reveal targets that can be inhibited to stabilize ELL2 for efficient BFEC formation. Together, the proposed studies may enable the development of novel adjunctive therapeutic strategies to specifically and efficiently eradicate latent reservoirs in HIV patients.

Public Health Relevance

Latent reservoirs of HIV are the principal impediment to eradication of infection as they harbor silent proviruses that resume active replication once therapy is disrupted. This proposal explores the potential of targeting our recently identified human cofactors that are important for HIV transcription to reactivate latent viruses, which can then be cleared by highly active antiretroviral treatment (HAART). The proposed studies may enable the development of novel adjunctive therapeutic strategies to specifically and efficiently eradicate latent HIV reservoirs in infected patients.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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NeuroAIDS and other End-Organ Diseases Study Section (NAED)
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Lawrence, Diane M
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University of California Berkeley
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