The Methylational Regulation Of HIV-1 Promoter By Apolipoprotein B MRNA Editing Enzyme Catalyzed Subgroup 3A(APOBEC3A)

Object: HIV-1 latent infection is one of the main reasons why AIDS cannot be cured.The key measure to cure AIDS is how to effectively activate and kill latently infected virus.The main factor in the formation and maintenance of the HIV-1 latent pool is CpG methylation in the HIV-1 5' LTR.APOBEC3A(A3A),a member of apolipoprotein B mRNA editing enzyme catalytic subunit 3(APOBEC3),can recognize methylated cytosine(methylcytosine,mC)and regulate DNA methylation.A3 A can effectively activate the latent virus,which was associated with its methylated cytosine deaminase activity and catalyze the methylated CpG of 5' LTR to demethylate.In order to determine the demethylation effect of APOBEC3A(A3A)on CpG island of HIV-1 5' LTR promoter,the methylated vector carrying the HIV-1 promoter was constructed and modified,and the eukaryotic vectors expressing A3 A isotype a and A3 A isotype b were constructed and selected,and A3 A mediated DNA-demethylation was further characterized and explorated its molecular mechanism which laied the foundation for the development of new HIV activators.Methods:1.The pLTR-Luc2P-EGFP was firstly constructed,which carried 5' LTR sequence of HIV-1 promoter.Then the methylation indicator plasmid pmeLTR-Luc2P-EGFP was modified by the M.SssI(CpG methylation enzyme)treatment and further verified through the bisulfite sequencing PCR and methylation sensitive restriction enzyme digestion.2.HIV-1 5' LTR contains eight CpG islands,which can be easily methylated.So the methylation sites of pmeLTR-Luc2P-EGFP were located in the plasmid promoter region,and the plasmid pme LTR-Luc2P-EGFP carried Luc2 P and EGFP indicator.Three methods were selected to detect methylation:(1)Western blot method to detect the protein EGFP expression level;(2)Luciferase method to detect relative luciferase activity and relative luciferase ratio;(3)Real-Time PCR method for Hpa II sensitivity analysis.For verifying that the three methylation detection methods initially selected were feasible,the methylated plasmid pmeLTR-Luc2P-EGFP and the unmethylated plasmid pLTR-Luc2P-EGFP were transfected respectively.And then the plasmids with different methylation levels(100%,75%,50%,25%,and 0%)were transfected to verify the abilities that three methylation assays could detect different methylation gradients.The results were further analyzed by Pearson correlation analysis using 0%,25%,50%,75%,100% as the standard value of the degree of methylation(100%,75%,50%,25%,0%),and the correlation between the measured values and the standard values of the three methods was evaluated.3.The cDNA of A3A-a and A3A-b were amplified from THP-1 mRNA and cloned into eukaryotic expression vectors pASIB-HA and pCAGGS-HA.The HEK-293 T cells were transfected,and the protein expression of A3A-a and A3A-b were detected by WB.The A3A-a and A3A-b highly expression vectors were screened.4.The A3A-a and A3A-b highly expression vectors and the negative control vector pASIB-HA-A3 G were co-transfected with the methylated promoter pmeLTR-Luc2P-EGFP into the HEK-293 T.To evaluate the demethylation regulation of A3A-a and A3A-b,the expression of EGFP was detected by WB,and the relative luciferase activity was detected by luciferase method.5.To study the de-methylation mechanism of A3 A,the A3 A large fragment plasmid was transfected into HEK-293 T cells,and the interaction with TDG was detected by Co-Immunoprecipitation.Results:1.The plasmid pLTR-Luc2P-EGFP carrying 5' LTR sequence of HIV-1 promoter was successfully constructed by double-enzyme and sequencing identification.Then the methylation indicator plasmid pme LTR-Luc2P-EGFP was successfully modified by the treatment of the M.SssI(CpG methylation enzyme)and further successfully verified through the bisulfite sequencing PCR and methylation sensitive restriction enzyme digestion.2.Methylation levels were detected by using WB,luciferase,and Real-Time PCR methods.Compared with the unmethylated plasmid p LTR-Luc2P-EGFP transfection group,in the methylation indicator vector pme LTR-Luc2P-EGFP transfection group:(1)EGFP protein expression was significantly reduced;(2)The relative luciferase activity was significantly weakened;(3)The sensitivity of Hpa II was significantly reduced.These results confirmed that the methylation indicator vector pme LTR-Luc2P-EGFP was effectively methylated and verified that the above three methods could be used for methylation detection.By transfection of plasmids with decreased degrees of methylation:(1)EGFP protein expression levels were gradually increased,(2)The relative luciferase activity and relative luciferase ratio were gradually increased,(3)The sensitivity of Hpa II was gradually increased.The Pearson correlation analysis showed that the above three methods had a strong positive correlation between the measured value and the standard value,and the Real-Time PCR method had the best correlation followed by the WB method,and the luciferase method was relatively minimal.These results demonstrated that the three methods could be used to detect different methylation levels.3.The results of PCR,enzymatic identification and sequencing showed that the A3 A eukaryotic expression vector pASIB-HA-A3A1-a/b and pCAGGS-HA-A3A2-a/b were successfully constructed.After screened by WB,A3 A proteins were highly expressed in pCAGGS-HA-A3A2-a and pCAGGS-HA-A3A2-b,which could be used in following experiments.4.The co-transfection experiment showed that in comparison with A3 G transfected control group,the expression level of EGFP and the relative luciferase activity in A3A-a and A3A-b groups were significantly increased.5.The Co-IP result showed that there were clear bands in the molecular weight of A3 A and TDG protein,which indicated that A3 A interacted with TDG.Conclusions:1.A3 A isotype a and isotype b could recognize the 5-methylcytosine and catalyze the demethylation of CpG island in HIV-1 promoter at the cellular level,which laid a function for further study on the role of A3 A in HIV latency infection.2.A3 A interacts with TDG,suggesting A3 A might participate 5-methylcytosine demethylation via TDG-dependent active DNA demethyation pathway.