The Role And Mechanism Of Interaction Between TIP60 And BRD4/FACT Complex In Regulating HIV-1 Latent Infection

AIDS,also known as Acquired Immune Deficiency Syndrome,is caused by the human immunodeficiency virus(HIV),which attacks the human immune system,causing human immune function defects,opportunistic infections,and secondary tumors,even seriously endangering human life,health,and safety.Since combination antiretroviral therapy(cART)(Cocktail therapy)was proposed in 1996 and widely used,AIDS has become a preventable and controllable chronic disease.CART combines three or more drugs to inhibit the replication of HIV,usually including nucleotides and non-nucleotides reverse transcriptase inhibitors(NRTIs and NNRTIs),protease inhibitors(PI),and integrase chain transfer inhibitors(INSTI),which can reduce the viremia in plasma to an undetectable level to control the damage of virus.However,due to the existence of HIV latent reservior in the human body,AIDS cannot be completely cured up till now.People Living with HIV(PLWH)must take medicine for life to prevent the rebound of viruses.The reasons for forming and maintaining the HIV latent reservior are very complex.At the early HIV infection,viruses can integrate into the DNA of the CD4+cells of host cells and become pre-viruses to participate in host cells’ life completing its replication and transcription.However,in this process,few pre-viruses of infected CD4+cells are in transcription silencing,which the immune system cannot recognize and clear.At the same time,cART drugs are also unable to kill pre-viruses that have been integrated into host DNA,thus,HIV cannot eradicate the human body.Once the external conditions are suitable,these latent viruses can be re-transcribed and re-translated,leading to the production of new viruses,which is the main reason for rebounding viremia and the great obstacle to HIV cure.Over recent years,"Shock and Kill" therapy has been adopted,which uses latency-reversing agents to induce the activation of HIV quiescent memory CD4+T,promote HIV transcription and replication,and combine cART to completely kill the active HIV.Meanwhile,active virion will cause cell pathological change and innate immune system-specific killing of cells,which greatly shortens the half-time of latent cells and leads to death rapidly,achieving the "function" cure of HIV.Lysine acetyltransferase 5(KAT5 or TIP60)belongs to the MYST family of histone acetyltransferases(HAT).The study recognizes that TIP60 can regulate and interact with the Tat protein.It can acetylate histone H4 on HIV-1 LTR to recruit BRD4 to LTR to competitively inhibit the binding of Tat protein a p-TEFb,which causes the latency of HIV.This study intends to explore whether TIP60 can regulate HIV latent infection by stabilizing BRD4 and whether BET inhibitors and other classic HIV latent reversal agents can regulate HIV latent infection through BRD4 and TIP60,providing a new target for finding HIV latent reversal agents.In the meanwhile,we identified a BET inhibitor,which has a certain reversal effect on HIV-1 latent infection cells.We intend to estimate its reversal effect and the mechanism on HIV-1 latent infection cells to provide a reference for the development of ideal latent reversal agents.First,after infecting HIV target cells,we estimated the change in TIP60 expression,discovering that HIV infection was able to significatively decrease the TIP60 expression of host cells.Knocking down or over-expressing TIP60,we found that it could inhibit the transcription and replication of 5’LTR,which negatively correlate with HIV replication.Following,reactivated HIV latent cells by classic HIV latent reversal agents,we found that the expression of TIP60 could down-regulate in a time-dependent manner detected by ELISA and Western Blot.After knocking down TIP60,the activation of HIV increased significantly,showing that it could promote HIV latency,and its deletion could promote HIV activation.Further research showed that the proteasome inhibitor MG 132 could not inhibit the degradation of TIP60 protein,while the Pan caspase inhibitor Z-VAD-FMK could effectively inhibit the degradation of TIP60,indicating that TIP60 might be degraded through the cysteinyl aspartate-specific proteinase pathway.By Co-IP detection,we found that TIP60 could bind with BRD4 of the BD1 structure domain.With knocking down HIV latent cells,the expression of the FACT complex decreased significantly.With knocking down SPT16,there were no significant changes in the expression of TIP60,which showed that SPT16 couldn’t opposite TIP60 expression regulation.At the same time,we found that a BET inhibitor Alobresib had a reversal effect on HIV latent cells and could regulate latent infection through the p-TEFb pathway,TIP60,and FACT.In summary,the combination of TIP60 and BRD4 could well stabilize BRD4 and jointly promote the incubation of HIV-1.In addition,TIP60 regulated the FACT complex and inhibited HIV-1 expression.These regulatory mechanisms provide new ideas for the development of new HIV-1 latent reversal agents.