The Influence Of L60V, I97L Mutant Core Protein Of Hepatitis B Virus On The HLA-A Expression And The Apoptosis In HepG2 Cells

BACKGROUNDHepatitis B virus (HBV) is a major human infectious pathogen.Many HBV carriers will develop chronic liver diseases, includingcirrhosis and primary hepatocellular cancer (PHC). The spectrum ofinfection ranges from fulminant hepatitis, acute self-limited hepatitis, andchronic hepatitis to asymptomatic carder (AsC) state. Studies have alsoshown that the immune response to HBV remains vigorous long after theepisode of acute HBV infection. Main effective cells to eliminate theHBV include CTL, Th and B cells. The cellular immune responsemediated by CTL is the most critical factor and plays a key role todetermine the clinical outcomes. The clinical outcomes of viral infectionresult from the interaction of viruses and their hosts. Over the past decade,increasing attention has been focused on the contribution of variant HBVstrains to the clinical outcomes of HBV infection, and some importantmutations, which display significant biological functions, have beenidentified, but the underlying mechanisms that are responsible for thediversity of clinical syndromes are not fully understood. It is generallyaccepted that HBV is not directly cytopathic for the infected hepatocyte.Instead, the hepatocellular damage and subsequent viral clearance ismediated by immune response. Core gene mutations are epidemiologicallyassociated with hepatitis activity. Several studies have reported that coregene mutations are more frequently detected in patients with fulminant orsevere hepatitis, but less so in patients with acute self-limited hepatitisand asymptomatic carriers. Thus, it has been suggested that mutations inthe core gene are significantly associated with active liver disease.Mounting evidences showed the hot-spot variations in hepatitis B viruscore gene included P5T, V13A, S35A, L59V, P68L, P130T and P135Qand, these common mutations were detected frequently in the HBV occultcarriers. The high frequency of L60V, I97L mutants in core region weredemonstrated in patients with active hepatitis, but the relationshipbetween these two mutant spots and the disease progress remains elusive. In a recent study, I97L, L60V and wild-type replication-competent HBVgenomes transferred into liver cells have different influence on thecellular expression of HLA-Ⅰ. Other evidences show these mutations inthe core gene might change the immune recognition sites of HBcAgthereby eliciting or evading immune clearance. I97L, L60V and wild-typeHBV strains have different influence on the cellular expression of HLA-Ⅰ.Some reports have demonstrated the influence of wild-type HBV strains,envelope protein and core protein on the apoptosis of HepG2 undergoingapoptosis induced by several cytotoxic agents. But compared with thewide-type core protein, whether core proteins with the hot-spot mutantshave the different influence on the cellular expression of HLA-Ⅰand theHepG2 undergoing apoptosis induced by some factors is still elusive. Wechose two hot-spot mutations in the C gene, L60V and I97L, to studytheir effect on the cellular expression of HL-A and the apoptosis incomparison with the wild-type core protein.AIMTo investigate whether the wide-type, L60V and I97L mutant HBcproteins could have different influence on the cellular expression ofHLA-Ⅰ; To study whether the wide-type, L60V and I97L mutant HBcproteins could have different influence on the HepG2 undergoingapoptosis induced by TNF-αand Act-D and further research the possiblesignaling pathway for them to inhibit the occurrence of apoptosis of theHepG2 cells.METHODS[1] The full-length HBV/C gene cDNA was obtained by PCR from atemplate (p3.8Ⅱplasmid containing 1.2 copies HBV gene) and a pair ofprimers were inserted into the enzyme cutting sites. After PCR, the PCRproduct was purified by gel extraction kit and the purified cDNA of HBcgene was cloned using general T-carrier pMD18-T to obtain clonedcarrier pMD18-HBWC, which was then transformed into DH5α, selected,amplified and then verified by PCR and the double enzyme cutting.[2] The construction of the wild-type HBc protein expressionvector(pEGFP-WT): pEGFP-C1 plasmid was digested by doubleenzymes and then purified by gel extraction kit. pMD18-HBV/C was also digested by double enzyme cutting and then HBV/C cDNA was obtainedand purified from pMD 18-T-HBVP22~e by enzyme cutting. We connectedHBV/C cDNA and pEGFP- by T4 ligase, then the product was clonedinto DH5α, and at last the positive clones were selected and verified byPCR, enzyme cutting and sequencing.[3] The construction of the L60V and I97L mutant HBc proteinexpression vector(pEGFP-V60 and pEGFP-97): We achieved these twovectors by site-mutagenesis on base of pEGFP-WT. Point mutations weremade in using QickChange site-directed mutagenesis kit. After PCR, themethylated parental DNA template without mutation was digested withDpnI restriction enzyme. Then the circular, nicked dsDNA wereTransformed into DH5αsupercompetent cells and repaired. The positiveclones transformed with the mutant plasmid were selected from the LBplates and subsequently the plasmid was extracted and verified by PCR,enzyme cutting and sequencing.[4] The transfection of the recombinant vectors: The eukaryoticexpression carriers, pEGFP-C1, pEGFP-WT, pEGFP-V60 andpEGFP-L97, were transfected into HepG2 cell strains by lipofectine2000respectively and then the positive cells were screened by G418.[5] The identification of the fusion EGFP-core proteins: Theexpression of the fusion EGFP-core proteins were verified usingfluorescence microscope observation and western-blot method. Thequantification was made by analytic software.[6] The detection of HLA-A mRNA and protein expression: TotalRNA was extracted by the rountine way and the HLA-A gene expressionwas detected by the reverse-transcriptase PCR. The products wereelectrophoresis by agrose and the results were quantified by the analyticsoftware.The HLA-A protein expression was measured by FlowCytometry (FCM).[7] Detection and induction of the apoptosis of the various HepG2cell strains: The stimulating duration is 16h, 32h and 48h, respectively.The proportion of cells containing sub-G1 DNA was examined by FlowCytometry (FCM) and propidium iodide (PI) staining. This proportion isused to represent the number of apoptotic cells. Laser Scanning Confocal Microscopy and Hoechst staining are carried out to observe the change ofthe nucleus of apoptotic cells in 32h.[8] The detection of the signal proteins in various groups: Wecollected the cells in 48h and detected the signaling proteins containingIKK-α,IκB-α. NF-κB P65,Caspase-8 and caspase-3 by western blotmethod.RESULTS[1] The wide-type HBV/C gene and clone carrier pMD18-HBV/Chave been obtained successfully, pEGFP-WT, containing the wide-typeHBV/C gene and enhanced green fluorescent protein gene, has been alsosuccessfully obtained, pEGFP-V60 and pEGFP-L97vectors aresuccessfully constructed by site-mutagenesis.[2] The G418 lowest lethal agent is 200μg/ml. The cells transfectedwith pEGFP-C1, pEGFP-WT, pEGFP-V60 and pEGFP-L97 vectors werethen selected using G418 at the concentration of 400μg/ml for two weeks,and the single clone selected showed the existence of the EGFP-fusionprotein by the fluorescence microscope observation respectively; and theexpression of the EGFP-wild-type core protein, L60V and I97L coreproteins, was detected by western blot. The quantitation of these threefusion proteins shows no marked difference(p＞0.05).[3] For the expression of the three fusion proteins, EGFP-wide-typecore protein, L60V and I97L core proteins detected by western blot, thereare no marked difference among these groups (p＞0.05)[4] The results of HLA-A mRNA and protein expression in variousgroups: In various groups, the results from RT-PCR show no expressionin the pEGFP-C1 group and obvious expression in the other groups.Compared with the pEGFP-WT group, The expression of HLA-A mRNAin the pEGFP-L97 one is markedly high (p＜0.05), but that in thepEGFP-V60 one is markedly low (p＜0.05). The results from FlowCytometry (FCM) show the same tendency.[5] The results of the apoptosis from various groups induced byTNF-αand Act-D: The results from flow cytometry and PI staining showthat the proportion of cell apoptosis in other three groups is markedly lowrespectively at various time points compared with the pEGFP-C1 group. In 16h, the proportion of cell apoptosis in the pEGFP-L97 group ismarked higher than that in the pEGFP-WT one. In 32h, the proportion ofcell apoptosis in the pEGFP-V60 group is marked higher than that in thepEGFP-WT one. In 48h, the proportion of cell apoptosis in thepEGFP-V60 group or pEGFP-L97 one is marked higher than that in thepEGFP-WT one respectively. The results from the control HepG2 cells,the pEGFP-C1 group, the pEGFP-L97 group and the pEGFP-V60group, which were not induced by any factors, show no difference in Ohand 48h.[6] The results of apoptotic detection by laser scanning confocalmicroscopy: The proportion of cell apoptosis in the pEGFP-L97 orpEGFP-V60 group is markedly high respectively compared with thepEGFP-WT group in 32h, but lower than that in the pEGFP-C1 one.[7] The results of the signaling proteins concerning the inducedapoptosis: The expression of NF-κb, IKK-α, IKB-αproteins are notmarkedly influenced in various groups, but caspase-3, 8 aredownregulated in the pEGFP-WT, pEGFP-L97 and pEGFP-V60 groupscompared with pEGFP-C1 one. The expression of caspase-3 andcaspase-8 is markedly low in the pEGFP-WT group in comparison withthe pEGFP-L97 and pEGFP-V60 ones.CONCLUSIONS[1] The expression vectors of wide-type core protein, L60V and I97Lmutant core proteins are established; The stable cell clones expressingnearly the same quantitation of the three fusion core proteins could beused to further research their functional difference.[2] The core proteins could upregulate the cellular expression ofHLA-A mRNA and protein. Compared with the wild-type core protein,The expression of HLA-A mRNA and protein in the I97L one is markedlyupregulated, but that in the L60V one is markedly downregulated and themechanism is still elusive.[3] The core proteins could downregulated the induced apoptosis ofHepG2 cells. The cell clones expressing mutant L60V, I97L core proteinsare prone to apoptosis compared with the one expressing the wild-typecore protein. [4] L60V or I97L core protein upregulates the cellular caspase-3 andcaspase-8 expression, which contributes to the upregulated apoptosis ofHepG2 cells transfected with L60V or I97L core protein.