The Study Of APOBEC3G Transcription Regulation Mechanism

BackgroundApolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G),the new member of cytosine deaminase family,is composed of 384 amino acid,with a molecular weight 46kD.APOBEC3G comprises two domains of cytosine deaminase,which can deaminate deoxycytidine(dC) residues to deoxyuridine(dU) in the growing minus-strand of viral DNA.APOBEC3G exert antiviral defense as an important factor of innate immunity.APOBEC3G is packed into virions by budding during the assembly of virions,and then enter the new infected cells with the viron to exert its antiviral effect either by catalyzing the deamination of dC to dU in the genome of retrovirus such as HIV and HBV or directly inhibiting the activity of reverse transcriptase independently of deaminase.APOBEC3G can be expressed in lung,liver,spleen,testiculus,ovary and granulocytes and lymphocytes of peripheral blood extensively,but the researches on the modulation of APOBEC3G transcription are limited.Current researches have focused on the regulation of APOBEC3G expression in hepatocytes and lymphocytes. Researches show that the phorbol ester can induce the expression of AOPBEC3G by the pathway of PKCα/βI/MEK/ER while the inducible expression of AOPBEC3G by IFNαmay be independent of STAT1.A variety of cytokines such as IFNγ,IL2,IL15,IL7 can also induce the transcription of AOPBEC3G..HCV can upregulate the expression of AOPBEC3G.by the stimulation of NS5A on the transcription of AOPBEC3G.Nuclear factor Sp1 and Sp3 can bind with the GC-box in the promoter of AOPBEC3G and participate the modulation of basic transcription of AOPBEC3G.. However the regulation of other nuclar factor and HBV on the transcription of AOPBEC3G.have not been reported at the present.Objective1.To investigate the modulation of USF1 gene on the transcription of APOBEC3G..2.To investigate the modulation of HBV on the the transcription of APOBEC3G.Methods1.In order to search for one or more potential binding sites of transcription factors,the on-line bio-software was used to analyze the full-length of the APOBEC3G promoter.2.Construction of pcDNA3.1(+)-USF1 vector:total RNA from HepG2 cells was isolated,reverse transcribed into cDNA.USF1 gene was amplified and then ligated with the pMD18-T vectors.The pMD-USF1 vector construction was identified by polymerase chain reaction by using the M13F and M13R primers pairs and further sequenced.Sequencing results alignment with UCSC database confirmed the target gene.The plasmid was extracted,and double digested by BamHⅠand XhoⅠ, subsequently ligated with the eukaryotic expression vector pcDNA3.1(+) to yield pcDNA3.1(+)-USF1 recombinant vector.After transfection into Huh7 cells for 48 hours,the total proteins were isolated,and the expression levels of the USF1 gene was identified by western blotting3.Effects of USF1 gene overxpression on the APOBEC3G:total RNA was extracted from transient transfected Huh7 cell lines for 48 hours with pcDNA3.1(+) and pcDNA3.1(+)-UF1 plasmids.The Real-time RT-PCR was performed to determine the mRNA of APOBEC3G.pcDNA3.1(+) and pcDNA3.1(+)-USF1 plasmids transfected in Huh7 cells,respectively.Selection of the stable cell lines was obtained by G418.Total RNA was isolated from transfected cells,and the mRNA of APOBEC3G was detected by Real-time RT-PCR.4.Effects of decreased USF1 gene expression on APOBEC3G:The short interfering RNAs(siRNAs) were synthesized,and transiently transfected into Huh7 cells.After transfection for 48 hours,the total proteins were extracted,and the USF1 protein expression was determined by western blotting to examine the interfering capacity of USF1 gene.Meanwhile,the total RNA was isolated,and the mRNA of APOBEC3G was detected by Real-time RT-PCR.5.The effect of USF1 on APOBEC3G promoter was determined by Dual-Luciferase(?)Reporter Assay System:pcDNA3.1(+) and pcDNA3.1(+)-UF1 were co-transfected with pGL3-A3G vector into Huh7 cells and HepG2 cells, respectively.In order to balance the transfection efficiency,phRL-SV40 plasmid as endogenous control,was added into the transfection.To be consistent with the number of molecules and mass of DNA transfected,salmon DNA was also added in the transfection as the supplement for quality control.Transfected cells were lyzed at 48-hour posttransfection,and dual luciferases enzymatic activity was detected to observe the effect of USF1 on APOBEC3G promoter in different cell lines.Dosage effect was investigated by transfecting different doses of pcDNA3.1(+)-USF1 plasmid and pGL-A3G into Huh7 cell under fixed total quality of DNA and pGL-A3G in transfection.6.Identification of the binding siteof the USF1 gene on APOBEC3G promoter: we analyzed the full-length of APOBEC3G promoter,obtained the position of E-box, and designed primers to amplify a series of 5'deletion fragemnts of APOBEC3G promoter from upstream transcription initiation site,including -1130,-795,-232,-159,-84 and -54.The sequences described previously were digested and inserted into the luciferase reporter vector,pGL3-basic,The target fragemnts were confirmed by comparing with the sequence in GenBank.And then,plasmids were extracted and co-transfeted with pcDNA3.1(+) or pcDNA3.1(+)-SUF1 plasmid into Huh7 cells, respectively.The activities of dual luciferases were determined to study the potential bingding-site of the USF1 protein on APOBEC3G promoter.7.The effects of USF1 E-box on APOBEC3G promoter by site-directed mutagenesis:the two nucleotide sites 91/-86 of E-box on APOBEC3G promoter were mutated by using the QuickChange site-directed mutagenesis kit,changing CAGCTG to CAATTG.After sequencing,the plasmid was extracted,digested and inserted into pGL3 -basicvector again to constructe recombinant pGL3-A3Gmut vector.The pGL3-A3Gmut vector was cotransfected with either pcDNA3.1(+) or pcDNA3.1(+)-USF1 plasmid.Meanwhile,wild type pGL3-A3G plasmid was also cotransfected with pcDNA3.1(+) or pcDNA3.1(+)-USF1 plasmid as positive control. Dual luciferases enzymatic activity was monitored to detect if USF1 could activate the luciferase expression of pGL3-A3Gmut vector after mutation.8.Impact of integral HBV particle on APOBEC3 G promoter:pUC 19-1.24HBV and pGL3-A3G vectors were co-transfected into Huh7 for 48 hours.,The transfected cells were lysed to determine the activities of dual luciferases.Therefore,we could observe if integral HBV particle has an effect on the activity of APOBEC3G promoter or not.9.The effects of HBV viral proteins on APOBEC3G promoter:the recombinant eukaryotic expression vectors of HBV large S antigen,core antigen or E antigen were co-transfected with pGL3-A3G vector into Huh7 cells.After 48 hours,the activities of dual luciferases were detected to study the potential effects of these viral antigens on the activity of APOBEC3G promoter.10.Expression of HBV P gene(RT/RnaseH domain) by Using the adenoviral expressing system:HBV/P(RT/RNAaseH) gene fragment was amplified from pU19-1.24HBV and inserted into pCMV-Tag2 vector,which was named as pCMV-tag-RT vector.Subsequently,,pCMV-tag-RT was amplified as the template, and HBV RT portion containing Flag tag was generated and double digested.The target gene was inserted into adenovirus shuttle vector,pShuttle-CMV,then transformed into the bacteria Bj5183,in order to recombinate with the adenovirus skeleton vector.The recombinant plasmid was transfected into Ad293 cells for packaging virus particles.After the tranfected cells were lyzed,the supernatant containing virus was collected to infect Huh7 cells.At 48 hours postinfection,the transcription of HBV/P gene(RT/RnaseH domain) was detected by reverse-transcript PCR.11.Expression of recombinant HBV polymerase RT domain by the baculovirus system:RT/RNaseH gene fragment was amplified by PCR from pUC19-1.24HBV plasmid,and inserted into the baculovirus shuttle vector pFastBacHT-RT.In order to generate recombinant Bacmid DNA,the recombinant vectors were transformed into DH10Bac.The recombinant plasmid was extracted and transfected into Sf9 cells,to generate recombinant baculovirus containing HBV P gene RT domain.The supernatant from recombinant baculoviurs was used to re-infect Sf9 cells for 48 hours, The infected cells were lyzed,and the expression was detected by western blotting using anti His antibody which recognizes His tag in the vectors.12.Effects of HBV P gene RT domain on APOBEC3G promoter:pCMV-tag-RT and pGL3-A3G vectors were co-transfected into Huh7 cells.Meanwhile,pCMV-tag and pGL3-A3G vectors were also co-transfected into Huh7 cells for 48 hours as negative control,.The transfected cells were harvested,and the activities of luciferase were detected.13.Statistic analysis:data were analyzed by the SPSS 13.0 software with one-way ANOVA,student's t test or LSD test,when P＜0.05 was regarded as significant.Result1.A number of potential transcription factor binding sites in APOBEC3G promoter including USF1 gene were identified.Therefore,we selected USF1 gene to study its function.2.Construction of pcDNA3.1(+)-USF1 vector:sequencing and alignment with UCSC database confirmed the sequence of target gene.Double digestion and PCR identified the pcDNA3.1(+)-USF1 vector was constructed successfully.A 34 KD protein displayed as a strip were observed obviously by western blotting in transfected Huh7 cells,which demonstrated the pcDNA3.1(+) -USF1 vector we constructed could express USF1 protein.3.Effects of USF1 gene overxpression on the APOBEC3G:the expressions of APOBEC3G mRNA in Huh7 cells with transiently transfected pcDNA3.1(+)-USF1 plasmid and stably expressed pcDNA3.1(+)-USF1 were appropriately twice and 1.7 times in pcDNA3.1(+) plasmid as control,respectively.4.Effects of decreased USF1 gene expression on APOBEC3G:the expression of USF1 in Huh7 cells which transiently transfected with siRNA was determined by western blotting was lower than control,but the expressions of GAPDH,which was regarded as endogenous control were similar in all groups.The expression of APOBEC3G was lower about 30%than the control group when detected by Real-time RT-PCR.5.The effect on APOBEC3G promoter by USFI:Dual-Luciferase(?)Reporter Assay results suggestedthat the luciferases enzymatic activities in Huh7 cells and HepG2 cells were about five times higher than the control that transfected blank plasmid(t＝-40.476,P＝0.000;t＝-42.960,P＝0.000).It suggested that USF1 could activate the APOBEC3G promoter in different hepatocytes.Simultaneously,the luciferases enzymatic activity increased accompany with the expression of USF1 enhanced,suggesting that USF1 gene expression upregulated may enhance the activity of APOBEC3G promoter(F＝204.750,P＝0.000).6.Identification of the site on APOBEC3G promoter binding to USF1 gene:we analyzed the full-length of APOBEC3G promoter,and found that there were four E-box sites in APOBEC3G promoter,the detail position including -1439/-1434,-757/-752,-288/-283 and -91/-86.A series of 5'deletion fragemnts of APOBEC3G promoter report gene were constructed successfully which were confirmed by PCR,double digestion and sequencing.The luciferases enzymatic activity detected in Huh7 cells after transfection suggested that the shorter in 5'deletion fragment,the weaker in activity of promoter.Compared to the control, the USF1 gene still could activate the expression of APOBEC3G when the APOBEC3G promoter was deleted to -159 position(t＝-96.058,P＝0.000).However, when the APOBEC3G promoter was deleted to -84 position,the luciferase enzymatic activity was similar to the control(t＝--1.482,P＝0.213).7.The effects of USF1 E-box on APOBEC3G promoter by site-directed mutagenesis:pGL3-A3Gmut vector was confirmed by sequencing that in position -91/-86,changing CAGCTG to CAATTG successfully.The following results of transfection into Huh7 cellssuggested USF1 gene had no activation on pGL3-A3Gmut vector,therefore,it demonstrated that USF1 gene could activate the transcription of APOBEC3G through CAGCTG in position -91/-86(F＝1833.717, P＜0.001).8.Impact of integral HBV particle on APOBEC3G promoter:the luciferase enzymatic activity in the control was four times compared to transfected group,when pUC 19-1.24HBV and pGL3-A3 G cotransfection(t＝-13.335,P＝0.000).it suggested that HBV inhibit the activity of APOBEC3G promoter.9.The effects of HBV viral proteins on APOBEC3G promoter:there were no significant difference in the luciferase enzymatic activity between the groups transfected with large S antigen,core antigen and E antigen(F＝2.878,P＝0.103). therefore,we thought that the activity of APOBEC3G promoter were not affected by different HBV viral antigens.10.Expression of HBV P gene(RT/RnaseH domain) by Using the adenoviral expressing system:HBV RT fragment was amplified and inserted into pCMV-Tag2 vector successfully.Subsequently,HBV RT fragment containing Flag tag was cloned into shuttle vector,and recombined with adenovirus skeleton plasmid successfully. The recombinant vector transfected into AD293 cells could package virus particle, when the transcription of HBV RT mRNA could be detected in infected Huh7 by RT-PCR.11.Expression of recombinant HBV polymerase RT domain by the baculovirus system::HBV RT fragment was amplified and inserted into pFastBacHT vector successfully.Subsequently,the vector recombined with baculorvirus skeleton plasmid in DH10Bac successfully,and transfected into Sf90 cells,which could package baculovirus particle.When the packaging viral particle re-infected Sf90 cells,western blotting dectection through anti-His antibody could be observed specific strip,which confirmed HBV RT domain could be expressed inbaculovirus system.12..Effects of HBV P gene RT domain on APOBEC3G promoter::the luciferase enzymatic activity after HBV P gene RT section and pGL3-A3G cotransfection was no difference compared to the control(t＝-0.404,P＝0.707),which suggested that HBV P gene RT section could not affect the activity of APOBEC3G promoter.Conclusion: 1.USF1 gene can regulate basal transcription regulation of the human APOBEC3G gene in hepatocyte,maybe the USF1 gene can bind at position -91/-86 of the APOBEC3G promoter to transactivate APOBEC3G transcription.2.HBV virus can inhibit the expression of APOBEC3G,but the large S antigen,core antigen,e antigen and HBV polymerase RT domain have no effect on the expression of APOBEC3G.