The Mechanisms Study Of Epigenetic Regulation Of Resveratrol-mediated HIV-1 Latency

Purpose We have developed the combined antiretroviral therapy(cART)to inhibit the human immunodeficiency virus-1(HIV-1)in infected patients effectively,with a number of researchers and doctors’ efforts.This therapy has successfully suppressed HIV-1 replication to undetectable clinical levels,thus helped the patients to reduce the mortality risk and tend to extend life.HIV-1 cannot be eradicate completely due to the existence of HIV-1 reservoir.HIV-1-infected individuals have to be treated with the cART lifelong to keep the low level of the viral load.The researchers tried many ways to solve the problems about how the HIV-1 reservoir to be thoroughly removed.The "shock and kill" is the most popular strategy to eradicate HIV-1 reservoir at present.Thus,it is a critical step to find effective reactivators-the "shock" agents.The latent HIV-1 reactivators should be safe,effective and with low toxicity,which are also the research priorities for the scientists.Resveratrol is a low molecular weight compound existing in many natural plants.It can be easily prepared,target many cellular candidates and diversity pathways.Previously,our lab has shown that Resveratrol could reactivate HIV-1 latency and inhibit inflammation cytokines.We subsequently performed experiments to test the HIV-1 latency reactivation regulations.In this study,we hope to find new targets to reactivate HIV-1 latency for Resveratrol,which will develop corresponding novel latency reversing agents.Methodology Initially,we conducted flow cytometry and luciferase reporter assay to verify the efficacy of different dose of Resveratrol in various J-Lat cells and TZM-bl cells.To ascertain that Resveratrol was safe,we performed multiple experiments to examine the potential toxicities in several HIV latency cell models and peripheral blood mononuclear cells(PBMCs).Resveratrol was co-treated with the conventional latency reversing agents to meet the requirement of the "shock and kill" strategy and the medicine appraisal.After confirming the effectiveness of Resveratrol activating HIV1 latency,we utilized multiple experiments to investigate Resveratrol mechanisms to facilitate HIV-1 latency,such as screening the different protein by LC-MS,identifying the contents of protein by Western blot analysis,and assaying the activity of the protein.Then we used siRNAs which knocked down target proteins to test whether it could increase the reactivation efficiency of HIV-1 latency.Results We used various latent HIV-1 cell models to detect the efficiency of Resveratrol and found it showed dose dependence.Then CCK8 experiments confirmed that concentration cytotoxicity 50%(CC50)of Resveratrol was safe and well tolerated in several HIV latency cell models and TZM-bl cells.Moreover,Resveratrol could raise the activation efficiency when co-treated with other latent HIV-1 reactivators.Several toxicity experiments confirmed the safety of Resveratrol in PBMCs.The results showed that Resveratrol did not cause the global T cells activation,not influence the release of cytokines and the proliferated abnormally of cells.Finally,we found the specific proteins(HDAC1 and KDM1)by conducting LC-MS experiments.Through western blot in J-Lat A2,we found that Resveratrol could enhance the methylation H3K4 by specifically inducing degradation of KDM1 protein.We also conducted several inhibition protein detection assays to confirm that Resveratrol has inhibition activities against HDAC1 and KDM1.Through siRNAs blocking the KDM1,we found that the depletion of KDM1 was able to reactive HIV-1 latency.Conclusions By analysis of above various results,we found that Resveratrol could reactivate HIV-1 latency and show obvious dose dependent.It has synergistic effect with others LRAs to reactive HIV-1 latency.Resveratrol did not induce the gold T cell activation or cause the release of cytokines.It did not generate the proliferated abnormally of cells.Thus,it is lower toxicity in cells.It utilized its epigenetic adaptor function on histone acetylation and methylation to promote HIV-1 expression.In summary,Resveratrol has the advantages of both low cytotoxicity and effective reactivating HIV-1 latency.It can also synergize with other LRAs to activate HIV-1 latency and maintaining enhanced epigenetic modifications on HIV-1 promoter.Resveratrol may be a good candidate of LRAs.