Although HAART is successful to block active replication of HIV-1 in AIDS patients, it does not eradicate viruses. Presence of latent HIV-1 reservoirs remains a major obstacle to the cure. Recently, it was uncovered that the size of latent HIV-1 reservoirs is much larger than previously estimated. Thus, identification of novel host genes that can be targeted for reverting HIV-1 latency is urgent. Our recent studies of host factors modulating HIV-1 replication identified that FACT (facilitates chromatin transcription) proteins, SUPT16H and SSRP1, restrict HIV-1 replication. Our further studies demonstrated that FACT proteins interfere with TAT-LTR transcriptional activities, and depletion of FACT proteins enhances HIV-1 transcription and facilitates reactivation of latent HIV-1. In this proposal, we wil explore molecular mechanisms how FACT proteins negatively regulate HIV-1 replication.
Aim 1 : Our genetic data showed that depletion of FACT proteins significantly enhance HIV-1 transcription. It was a surprising result considering that FACT proteins generally facilitate transcription. It is critical to study FACT's function under circumstance of HIV-1 infection, which will provide new knowledge about FACT functions that might be unique for HIV-1. We postulate two possible mechanisms that might lead to FACT suppressive activities: (i). Presence of FACT proteins might affect P- TEFb activity in HIV-1 transcriptional elongation; (ii). FACT proteins might process altered nucleosome exchange activity for HIV-1 transcription. These hypotheses will be thoroughly tested by a series of experiments using multidisciplinary approaches.
Aim 2 : Our preliminary results indicated that SUPT16H might directly bind with HIV-1 TAT and recruit to LTR promoter. However, a lot of information still lacks for further characterization of these interactions. Thus, we will study the molecular details of FACT interactions with HIV-1 TAT-LTR as well as other key host transcriptional factors (PAF1 and P-TEFb). We will (i) map which domain(s) of SUPT16H mediate its direct interaction with TAT; (ii) determine whether LTR recruitment of SUPT16H depends on TAT or PAF1; (iii) evaluate the impact of FACT proteins on P-TEFb and TAT-LTR interactions.
Aim 3 : If indeed FACT proteins impose an inhibitory effect on HIV-1 transcription, through which they might play a role in regulating HIV-1 latency. Our earlier results using HIV-1 latently infected J-LAT cells confirmed that depletion of FACT proteins by RNAi spontaneously reverts HIV-1 latency. In this aim, we will further evaluate the effects of FACT proteins on HIV-1 latency in a primary CD4+ T cell model: (i) Role of FACT proteins in suppressing HIV-1 transcription for latency establishment will be determined; (ii) Effects of FACT protein depletion on HIV-1 reactivation from latency will be measured. (iii) Coordination of FACT proteins with other HIV-1 transcriptional suppressors, histone deacetylases (HDACs), will be investigated. Above all, we expect that our in-depth functional studies of FACT proteins will shed light in understanding their roles in HIV-1 replication and provide sufficient scientific foundation to target them for HIV-1 anti-latency therapy.
Although the antiretroviral therapy is efficient to treat HIV/AIDS and slow the course of the disease, the presence of HIV latent reservoirs hinders the viral elimination and there is still no cure. We propose to dissect the molecular mechanisms of nucleosome chaperone proteins, SUPT16H and SSRP1 forming the FACT (facilitates chromatin transcription) complex, in suppression of HIV transcription. We will further explore the role of FACT proteins in HIV latency using a primary T cell model, and test whether their depletion might benefit efficient HIV reactivation as the initial step for purging HIV latent reservoirs.
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|Huang, Huachao; Santoso, Netty; Power, Derek et al. (2015) FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency. J Biol Chem 290:27297-310|