Molecular Mechanism Of APOBEC3A On The Regulation Of HIV-1 Promoter Demethylation

Objective: Acquired Immune Deficiency Syndrome(AIDS)caused by Human Immunodeficiency Virus(HIV-1)is one of the most important public health problems around the world.At present,antiretroviral drugs are the main method of controlling HIV-1,but due to the existence of viral latent pools,these drugs fail to completely cure AIDS.Studies have shown that the existence and maintenance of HIV-1 latent pool is associated with cytosine methylation in the CpG islands of the HIV-1 promoter 5'LTR.Our previous study demonstrated that the host restriction factor APOBEC3A(A3A)catalyzed the demethylation of cytosine in the CpG islands of the HIV-1 promoter,and promoted downstream protein expression.However,the molecular mechanism of A3 A catalyzing DNA demethylation remains to be further explored.A3 A,as a cytosine deaminase,recognizes methylated cytosine(mC),turns it to be thymine through deaminating it to form thy mine,resulting in a T:G mismatch.We hypothesize that this base mismatch activates the downstream base excision repair(BER)pathway to excise and repair T:G mismatch,and re-converts mC to cytosine(C),thereby completing the demethylation of mC.To prove this hypothesis,and uncover the molecular mechanism of A3 A involved in active DNA demethylation,in this study,the elements that play key roles in the BER pathway,including DNA glycosylation enzymes: methyl-CpG domain protein 4(MBD4)and thy mine DNA glycosylase(TDG),Gadd45 a,were analyzed as follows.Methods: 1.TDG,MBD4 knockdown and non-knockdown 293 T cell lines(shTDG-293 T,shMBD4-293 T,shNC-293T)were constructed,respectively.Then,pCAGGS-HA-A3A-a(A3A)or pCAGGS-HA-A3G(A3G,control)with methylated pmeLTR-Luc2P-EGFP or unmethylated pLTR-Luc2P-EGFP reporter plasmid were cotransfected into shTDG-293 T,shMBD4-293 T and shNC-293 T cells,respectively.The relative activity of luciferase was used to determine the effect of TDG and MBD4 knockdown on the demethylation of A3 A.2.Recombinant plasmids pCMV-Myc-TDG(TDG)expressing TDG and pCMV-Myc-MBD4(MBD4)expressing MBD4 were constructed,respectively.Following,TDG or MBD4 was co-transfected with A3 A into 293 T cells.The bindings of A3 A with TDG,A3 A with MBD4 were detected by immunoprecipitation assay,and the interaction between A3 A and TDG or MBD4 was detected by Western blotting.3.The recombinant plasmid pCAAGGS-Flag-Gadd45a(Gadd45a)expressing Gadd45 a was constructed,and then Gadd45 a,A3A and TDG,or Gadd45 a,A3A and MBD4 were co-transfected into 293 T cells.The bindings of A3 A with TDG,A3 A with MBD4 were detected by immunoprecipitation assay in the presence of Gadd45 a,and the role of Gadd45 a protein in the interaction between A3 A and DNA glycosylase was explored.At the same time,Gadd45 a was co-transfected with TDG or MBD4 into 293 T cells,and the interaction between Gadd45 a and DNA glycosylase was also analyzed.Results: 1.The morphology of TDG and MBD4 knockdown cells did not differ from the negative control cells.Western blotting confirmed that the expression of the corresponding protein in TDG and MBD4 knockdown cells was significantly reduced.Compared with shNC-293 T cells,the relative activities of luciferase in shTDG-293 T and shMBD4-293 T cells co-transfected with A3 A and pmeLTR-Luc2P-EGFP was decreased.In contrast,there was not significantly changed in the relative activity of luciferase in TDG and MBD4 knockdown cells co-transfected with A3 G and pmeLTRLuc2P-EGFP.2.The recombinant plasmids pCMV-Myc-TDG and pCMV-MycMBD4 were digested by EcoR I and Kpn I.The electrophoresis showed that the band size was consistent with the expected size,and the sequencing result was also consistent with the expected result.And then A3 A was co-transfected with TDG or MBD4 into 293 T cells,and immunoprecipitation with anti-Myc antibody detected the interaction between A3 A and Myc-TDG or A3 A and Myc-MBD4.3.The recombinant plasmid pCAAGGS-Flag-Gadd45 a was identified by Kpn I and Xba I double enzyme digestion,and the electrophoresis showed that the band size was consistent with the expected size,and the sequencing result was also consistent with the expected result.The 293 T cell lysate co-transfected with Gadd45 a,A3A and TDG or MBD4 was immunoprecipitated with the anti-Myc antibody,and the immunoblotting results showed that the interaction of the A3 A protein with Myc-TDG and Myc-MBD4 increased when the Gadd45 a protein was co-transfected.And Gadd45 a can also be co-precipitated with TDG or MBD4 protein.Conclusions: 1.The knockdown of TDG and MBD4 can attenuate A3 A demethylation activity,suggesting that TDG and MBD4 are involved in A3A-mediated demethylation of the HIV-1 promoter.2.A3 A interacts with the downstream molecules of the BER pathway,TDG and MBD4.3.Gadd45 a molecule enhances the interaction of A3 A with TDG and MBD4,and Gadd45 a also interacts with TDG and MBD4.Our results confirm that A3 A is involved in active DNA demethylation via the BER pathway.