Mechanism For Regulation Of HLA-I Expression On HepG2 Cells With HBV Wild Type And Nucleocapsid Mutants

To study the HLA-I/antigen peptide complex expression on HepG2 cells and to explore the mechanism of expression by HBV wild type and nucleocapsid mutants(L97, V60)To construct HBV genome wild type and core mutant stable expression vectors for experimental investigation. The site-directed mutagenesis was performed to introduce specific core point mutations L97 and V60 into 1.2 copies of HBV genome plasmid p3.8 II. Sequence analysis of constructed mutant plasmids p3.8 L97 and p3.8 V60 demonstrated mutations at nt 2189 A - C and nt2078 6- G respectively. The Southern blots for HBV DNA of HepG2 cells transfected with plasmids showed 3.8 kb hybridized bands clearly. The HBV antigens in their culture supertants were detected with ELISA positively. The plasmid p3.8II and mutant plasmid contructs were subcloned respectively into EB virus based vector EBO-plpp for stable expression. The vector constructs EBO-wild type(WT), EBO-L97 and EBO-V60 were reconfirmed by restriction enzyme digestion and DNA sequencing.To investigate the characters of HLA-I expression on HepGa cells by HBV wild type and nucleocapsid mutants L97,V60. EBO vector contructs were transfected into HepG2 cells respectively and HBV antigen in their culture supernatants was quantified by ELISA. The value of HBeAg s/co of EBO-wild type was much higher than that of mutant EBO-V60 andEBO-L97, while the values of HBsAg S/N of three constructs were in similarlevel, indicating similar transfection rate in the experiment. HLA-1 expression on HepG2 cells were analysized by flow-cytometry. Fluorescence intensity of HLA-1 expression of transfected cells by EBO empty vector was in low level(2.2). It was elevated by EBO-WT(18.2), while that of L97 increased to 34.5 and V60 declined to 3.4. It indicated that HBV might enhance the expression of HLA-1/antigen peptide complex on HepGa cells. Hot-spot mutations of HBV nucleocapsid protein L97 and V60 could influence the expression level of HLA-1 on host cells.To study the basic mechanism for up-regulation of HLA-1 surface expression on HepG2 cells by HBV. Compared with the result of EBO vector, the RT-PCR product bands of HLA-A cDNA and the Western blot bands of HLA-1 protein from HepG2 cells transfected with HBV constructs were identified clearly. But the intensify of both bands decreased successively as EBO-L97, EBO-WT and EBO-V60. HBV transfected cells revealed significant increase in the expression of TAPi gene mRNA than EBO vector and showed similar level among 3 constructs. The expression of LM?2 and Tapasin genes transcripts did not be detected in all samples. The results indicated that the enchancement of HLA-I heavy chain synthesis and TAPi gene mRNA expression in HBV transfected cells might mediate the up-regulation of HLA-I surface expression. The RT-PCR product bands of TNF- a gene from EBO-WT and EBO-L97 transfected cells were identified clearly and in same level, while IFN- P gene expression did not be detected. In comparision of the results from anti-TNF- a monoclonal antibody blocked cells with untrealed EBO-WT and EBO-L97 host cells, HLA-I fluorescence intensity failed from 7.73 to 3.89 and 12.26 to 6.21 respectively, the intensity levels of HLA-A and TAPi cDNA bands also reduced. It indicated that HBVenchancement the expression of HLA-I on HepG2 cells was partially dependent on the autocrine effect of TNF- a produced in the cells. TNF- a could up-regulate HLA-A gene and TAP1 gene expression at transcriptional level.