The Mechanism Of HCV E2 Protein Promoting Activation And Proliferation Of B Lymphocyte And Immunogenic Analysis Of E2 Mutant

Hepatitis C virus is an enveloped virus with a positive-sense single-standed RNA genome. HCV is a major factor of post-transfusion and community-acquired hepatitis in the world and causes a variety of liver diseases, including acute and chronic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma. Except causing liver pathological changes, HCV infection may induce extra-hepatic immune related manifestations in high percentage of infected patients.Immune system related manifestations include a myriad of conditions ranging from sub-clinical cryoglobulinemia to overt lymphoproliferative malignancy. Over 170 million people worldwide are infected with HCV, 80% of these develop chronic hepatitis C. HCV infection is associated with HCV gene variation, antagonistic reaction to immunity system of host cell and relative low levels of expression and replication of HCV. Recent studies show that the binding of HCV enveloped E2 protein to CD81 molecule leads to the lymphocyte functional disorders, which may play an important role in HCV chronic infection and have closed relation with B cell lymphadenoma and cryoglobulinemia. Associated with formation of HCV infection is hard to induce neutralizing antibody and thus leading to chronic infection. The interaction of HCV enveloped E2 protein with CD81 molecule has been considered to be related with this situation.1. The interaction of HCV envelope E2 protein with CD81 promotes activation and proliferation of B lymphocytesThe binding of HCV envelope E2 protein with CD81 molecule on the surface of B lymphocyte alters the normal signal transductional pathway of B lymphocyte activation, which may lead to nonspecific activation of B cell. We transfected 293T cell with plasmid expressing E2 protein to producing E2 protein. The expressed E2 protein was conjugated to ELISA microplate which was precoated with anti-E2 mAb H53. Then Raji cells were seeded onto the microplate and cultured . The condition of adherent growth of cells was observed. The recombinant lentivirus of human CD81 siRNA wan constructed and infected the Raji cells and down-regulated CD81 expression on the surface of Raji cells. The same experiment was repeated in the cell and the state of adherent growth wasn't viewed. These results showed that the expressing E2 protein was be able to interact with CD81 molecule on the surface of Raji cells. We prepared immobilized HCV envelop E2 protein and then stimulated Raji cells with the immobilized E2 protein and soluble E2 protein,respectively. The level of phosphorate-tyrosine of the proteins in Raji cells was detected by western blot. The results indicated that immobilized E2 protein remarkably raises the level of phosphorate-tyrosine in Raji cells , but the soluble E2 protein hasn't obvious impact to phosphorylation of tyrosine of Raji cell. It was presumed that immobilized E2 protein was resembled to natural viron enveloped E2 protein and may more efficiently interact with CD81 molecule. The Raji cells , CD81 expression silenced, was stimulated by immobilized E2 protein.The level of phosphorate-tyrosine of Raji cell didn't change, which illustrates that E2 protein is via CD81 molecule to regulates the signal pathway of Raji cell. The expression of Bcl-2 protein and Bax protein in Raji cell, which were apoptosis-associated proteins, were analized by western blott when the Raji cells were stimulated by immobilized E2 protein. The results showed that expression level of Bcl-2 protein was decreased, thus Bax protein increased, which indicated that HCV E2 protein had important contribution to promote Raji cell proliferation. The CD80,CD86 and CD21 molecules on the surface of Raji cell, which were related proteins with the activation of B lymphocyte, were detected by Flow cytometric analysis. The results show that the expression of CD80 and CD86 molecules were elevated and the CD21 was decreased.These indicated that E2 protein probrably activated the Raji cell and promoted proliferation of the Raji cell.2. Immunogenic analysis of mutational HCV E2 proteinHCV E2 protein induces phosphorylation of tyrosine in B lymphocyte and promotes proliferation of B lymphocyte by up-regulating expression of anti-apoptosis protein Bcl-2 and down-regulating expression of enhancing apoptosis protein Bax. HCV E2 protein also increases the expression of activated molecule of B lymphocyte. These results indicate that E2 protein may activate signal transduction pathway and initiate a series of biological effect of B lymphocyte. The activation of B cell by E2 protein make it difficulty to induce the neutralizing antibody after HCV infection. So ,we presume that elimination of the linked activity of E2 protein with CD81 would be profited to producing of neutralizing antibody.Plasmids expressing mutant of HCV E2 protein of H77 strain were constructed, in which the codons encoding the aa442 Phe(F) and the aa529 Trp(W) of E2 protein were both changed into the codon encoding Ala(A). HEK293T cells were transfected with the two mutant plasmids E2-W529/A and E2-W442/A and then the culture supernatant and cell lysate were analyzed by E2 polyclonal antibody.The results showed that the mutation didn't affect the expression and secretion of E2 proteins. We analyzed the binding by ELISA between the two mutans and the fusion protein Trx (thioredoxin)-CD81 (LEL, large extracellular loop)expressed by E. coli. The results showed that the mutants lost the binding function with CD81 LEL.The analysis of FACS showed that the two mutants were also no longer binding with the CD81 on the surface of cell. The two plasmids were transfected to HEK293T cells and then analysed the conformation of the two mutants with E2 conformation-dependent monoclonal antibody by immunofluorescent technique(IF). The results showed that the mutation of the two sits didn't affect the conformation of E2 protein. The two mutants were immobilized and then to stimulate the Raji cell. There were no obvious changes about the the level of protein phosphorate-tyrosine in Raji cells, which indicated that elimination of binding activity of E2 protein with CD81 molecule deprivated the activation function of E2 proein to the signal transduction pathway in Raji cell.The aa442 and aa529 of HCV E2 protein located respectively in two linear epitopes of B cell. The epitope including aa442 has neutralizing activity and the other including aa529 has no obvious neutralizing activity. For keeping more epitopes of neutralizing antibody,we chose the plasmids of mutant E2-W529/A and wide-type E2 to immune BALB/c mice. Blood samples were collected regularly by retro-orbital puncture from immunized mice.The antibody kinetics of the mice serum were analysed by using wide E2 and mutational E2 proteins as antigen. The results showed that the level of serum antibody kinetics of these mice immuned with mutative E2 protein were similar to the wide E2 ,which indicated that the mutation of W529 didn't affect the immunogenicity of E2 protein. The amino acid residue APTYSWGA(aa524-aa531) was a linear epitope of HCV. The antibody of the epitope may inhibit the binding of E2 protein with CD81.We constructed expression plasmid fusing the red fluorescent protein(RFP)into the C-termination of the epitope and then analyzed the reaction between this fused epitope and mouse serum by IF after transfecting the 293T cell. The results indicated that the mutation of W529A decreased the antigenicity of the epitope. The epitope AP33 , amino acid residue QLINTNGSWHIN (aa412-aa423) in HCV E2 protein, was also a linear epitope of B cell, which was high conservation in the six geneotypes of HCV and may effectively neutralize the infection of HCV. We fused the RFP with the epitope and analyzed the antibody response of mice serum by IF. The result showed the mutation of W529/A didn't influence the epitope antigencity and both wide and mutant E2 may induce the antibody to the epitope. We transfected the 293T cell with plasmids of expressing 11 strains HCV E2 proteins including 6 genotypes and detected the cross-reaction of mice serum. Results: the mutant E2-W529/A immuned mice serum has cross-reaction with these 11 strains HCV E2 proteins. For analyzing the neutralizing effects of mouse serum, we constructed 8 strains HCVpp and the 8 strains HCV were from different countries and involved 4 genotypes. The data showed that the infection of the 8 strains HCVpp to Huh7.5 cell were effectively neutralized by the wide-type E2 and mutant E2-W529/A. The neutralizing effects of the both to the same strain HCVpp were parallel. Except using the HCVpp, We also evaluated the neutralizing effects of these mice serum with JFH-1 HCVcc . The infection of HCVcc could be inhibited by the both immuned mouse serum and the inhibition efficiency was similar.Summary:1. In this study ,we stimulated Raji cells with immobilized HCV envelope E2 protein, which induced the phosphorylation of the protein tyrosine in Raji cells ,up-regulated the expression of Bcl-2 protein and down-regulated the expresse of Bax protein. The expression of CD80 and CD86 molecules expressed on the surface of Raji cells was increased and the CD21 was decreased.These changes were inhibited in the Raji cells on which CD81 expression was silenced. The results indicated that HCV E2 protein may promote the proliferation and nonspecific activation of B lymphocyte via CD81 molecule.2. According to this study, we constructed an E2 protein mutant by changing the aa529 Trp(W) into Ala. The E2-W529/A mutant didn't affect the conformation of E2 protein. For elimination of the binding activity with CD81, the mutant was no longer inducing the phosphorylation of the protein tyrosine and regulating the expression of apoptosis-associated proteins. We used the plasmids E2-W529/A to immunize the mice and detected the antibody positive rate up to 100% after the first immunization. The antigenicity of the E2 protein mutant was similar with wide-type E2 protein and may induce effective HCV cross-reaction neutralizing antibody. This mutant may represent a strategy for effective HCV vaccine development.