The Effect Of LEDGF/p75 In HIV Latency And Proviral Transcriptional Reactivation

In recent decades,acquired immunodeficiency syndrome(AIDS)caused by human immunodeficiency virus(HIV)infection has been a serious global health threat.Although WHO has adopted various measures to control the spread of AIDS,and statistical results show that the prevalence rate was stabilized and the incidence rate was decreased,the mortality rate related to AIDS has risen sharply in recent years.This epidemic is still a serious public hygiene issue.Antiretroviral therapy(ART)can inhibit virus replication and reduce the virus titer in the blood of patients.However,viral genes persist in the latent cell pool,especially CD4~+T cells.Once patients stop taking medicine,external stimuli will again induce the transcription and translation of the latent viral genome to generate new viruses.These latent cell banks are established in the early stages of infection,can survive for a long time and produce new viruses,which has become a major obstacle to HIV eradication.Finding and eliminating cells latently infected by HIV is the ultimate goal of HIV-related research.So far,there are two treatment strategies:1."shock and kill".A strategy aims to reactivate the transcription of the provirus,the patient’s immune system can thereby eliminate the infected cells.2."block and lock".A strategy designed to completely inhibit the transcription of the provirus and make the latent cell bank permanently in a deep dormant state.A deep understanding of the regulatory mechanisms of HIV latency and reactivation is necessary,no matter which strategy was adopted.The process of latency to activation of HIV-infected cells is mainly embodied as from transcriptional arrest to transcriptional activation at the molecular level.Considering a wide range of transcription factors may regulate the silencing and activation of latent HIV genome during the transcription process,the specific regulatory mechanism remains to be further studied.Recent studies have revealed that Lens epithelium-derived growth factor(LEDGF)/p75 can bind to Iws1 and Spt6 to repress proviral transcription in latently infected cells.LEDGF/p75 was initially recognized as a chromatin-related factor,LEDGF/p75 integrases binding domain(IBD)renders LEDGF/p75 serves different roles depending on the cellular partners it binds.Given all this,LEDGF/p75 may have more functions in HIV transcription regulation,the regulatory molecular mechanisms of LEDGF/p75 in proviral transcription remain to be further explained to provide a new strategy for the complete cure of HIV.Methods1.To prove the regulation of LEDGF/p75 on HIV latency,we knocked down LEDGF/p75 in HIV latently infected cell line,and performed flow cytometry tests on the 3rd,6th,and 9th days to detect HIV transcriptional activation.To prove the function of LEDGF/p75 in transcriptional reactivation,we performed the same experiment with TNFα,and detect the transcriptional activation status of HIV at 0 h,4 h,and 8 h after stimulation by flow cytometry experiments.2.To screen the protein that binds LEDGF/p75 for in-depth study,we overexpressed exogenous LEDGF/p75 in mammalian cells,performed protein affinity purification.We performed mass spectrometric identification for the obtained protein with acetone precipitation and identified LEDGF/p75 binding protein.3.To investigate the molecular mechanism of the regulation of LEDGF/p75 and LEDGF/p75 combined transcription factors in HIV transcription,we applied the luciferase reporter assay,HIV latency model cells,ChIP(chromatin immunoprecipitationexperiment).These experiments were conducted to explore the specific mechanism of LEDGF/p75 on HIV transcriptional regulation.This research aims to reveal the molecular mechanism of LEDGF/p75 in the process of transcriptional silencing and transcriptional reactivation,explore the possible intervention of small molecule compounds targeting these molecular mechanisms on HIV latent and reactivation.Results1.LEDGF/p75 can inhibit HIV transcriptional activation and promote HIV latency,while can promote HIV transcription reactivation in HIV transcription elongation.2.RNA Polymerase Ⅱ-associated factor 1(PAF1)complex interacts with LEDGF/p75 and promotes the establishment and maintenance of HIV latency.3.LEDGF/p75 recruits PAF1 complex among the LTR(Long Terminal Repeated)region of HIV,the PAF1 complex plays a role in blocking the elongation of RNA Pol Ⅱ(RNA polymerase Ⅱ),thereby promote HIV latency.4.LEDGF/p75 and HIV LTR and PAF1 complex dissociate in the process of latent HIV transcriptional reactivation,at the same time,MLL1(Myeloid/Lymphoid Or Mixed-Lineage Leukemia Protein1)/COMPASS complex(Complex Proteins Associated with Set1)was recruited and H3K4me3 modification is established in the LTR region,thereby the combination of SEC(Super Elongation Complex)and LTR was promoted,and the pausing RNA Pol Ⅱ was released,and the transcription of viral genes was activated then.5.CKⅡ(Casein Kinase Ⅱ)catalyzes the phosphorylation of serine-136,serine-142,serine-153 in the MLL1 motif IBM(Integrase Binding Motif),thereby enhanced the of MLL1 competing PAF1 to bind LEDGF/p75.6.CKⅡ plays an important role in the reactivation of latent HIV transcription.Inhibiting its kinase activity can prevent the transcriptional activation of latent HIV in human primary CD4~+T cells.ConclusionOur research suggested the dual function of LEDGF/p75 in viral gene transcription during HIV incubation and activation.During latency,LEDGF/p75promotes promoter-proximal pausing of RNA polymerase Ⅱ(RNA Pol Ⅱ)by recruiting the PAF1 complex to the provirus.During latency reversal,CKⅡ phosphorylation modifies serine-136,serine-142,serine-153 of MLL1 IBM,enhancing the binding affinity of MLL1/COMPASS complex with LEDGF/p75.The phosphorylated modified MLL1/COMPASS complex competitively binds to LEDGF/p75 and HIV LRT,dissociates the PAF1 complex from LEDGF/p75 and HIV LTR.The MLL1/COMPASS complex catalyzes the modification of H3K4me3,promotes the recruitment of SEC complex among LTR and releases the pausing RNA Pol Ⅱ to enhance the transcription of viral genes.Our findings suggest that inhibiting the activation of CKⅡ or inhibiting the combination of LEDGF/p75 and PAF1 may provide a new strategy to completely HIV latent infection.