The Effect Of HCV Envelope Protein Adaptive Mutations On Virus Infectivity And Antibody Neutralization

Hepatitis C virus(HCV)is a single strand RNA virus with enveloped membrane,which is an important pathogenic factor causing human liver disease.Currently,there are about 120 to 170 million HCV infections in the world,The incidence of HCV infection is higher in Africa than in Europe and America.HCV is mainly transmitted by blood,which causes liver diseases such as hepatitis,liver fat degeneration,cirrhosis and liver cancer,at the same time,it is also highly correlated with diabetes,lymphoma and other extrahepatic diseases.Chronic infection is an important characteristic of HCV infection,and the infected patients have no obvious symptoms in the early stage of HCV infection,and about 80%of the infected patients develop chronic infection before they develop clinical manifestations of liver disease.Chronic infection of HCV is the main pathogenic factor of liver cirrhosis and liver cancer in developed countries such as Europe,America and Japan.Since has been found in 1989,the molecular biology of HCV,pathogenic mechanism,drug and vaccine research have attracted global attention.The development of HCV replicon system and cell culture-derived HCV(HCVcc)is an important milestone in the history of HCV research.Research on HCV cell infection model provides an important tool for developing anti-HCV drugs.In recent years,the new generation of direct anti-HCVdrugs(DAAs)has made HCV becomes a curable disease.Eradicate of HCV infection is still depends on effective vaccine,HCV and HIV vaccine development experience similar difficulties,despite a series of different types of HCV vaccine in the process,some of the vaccine has terminated the process,and there is no vaccine shows a good application prospect.Lacking checking mechanism in the process of HCV genome duplication guided by HCV RNA polymerase,HCV genome is highly variable.Therefore,HCV quasispecies exists in the patients.According to the heterogeneity of the genome,HCV is divided into seven different genotypes,each of which can be divided into dozens of subtypes.The high variation of HCV genome is an important reason for its high incidence of chronic infection and the utmost obstacle to vaccine development.Our country belongs to the HCV medium epidemic area,the number of HCV infected people was 30-40 million.The HCV infection in Chinese patients is dominated by lb and 2a,but in the pearl river delta and the southwest,there are more common of type 3 and type 6 infection.HCV envelope protein formed by E1 and E2 glycoprotein of heterologous dimers and mediating cell invasion process,E1 and E2 are highly glycosylation protein,the function of E2 protein is clear.It is the main protein which mediates the sequential action of HCV and multiple receptors on the surface of hepatocytes.Cross-neutralizing antibodies against each genotype can be detected in the serum of HCV infected patients,which are mainly targeted on E2 protein.After the virus absorbed into the cell,the fusion of the viral envelope and the endosomal membrane may be triggered by El protein.However,the accurate function of E1 protein remains to be further studied.There are few reports of neutralizing antibodies against El protein.At present,it is not possible to isolate HCV directly from the infected patients,HCVcc is the most natural model to study HCV infection mechanism at the cellular level,which is similar to virus particles in serum of HCV patients in many aspects including invasion of host cells,infection characteristics and virus life cycle.On one hand,virus particle binding lipoprotein helps to capture molecules such as heparin and low-density lipoprotein receptors on the surface of hepatocytes to help the virus accumulate on the surface of hepatocytes,on the other hand,lipoprotein can hide the HCV envelope protein neutralizing epitope,thereby facilitating HCV evade humoral immune response.HCVcc,which is widely used by researchers,contains the non-structural genes of 2a JFH1 strain,which can be obtained by replacing different type/plant structure genes with different HCVcc.Which can be produced and released into the cell culture by transfection of HCV genomic RNA to Huh7 or Huh7.5.1 cells.In our previous experiments,it was found that the neutralization sensitivity of the original J6/JFH1 HCVcc produced by electroporation was significantly different from the HCVcc which obtained from successive culture.Based on this phenomenon,we studied the effects of envelope protein mutations from HCVcc successive culture on the virus infectivity.On the other hand,this study detected the neutralization activity in the serum of an aggregated HCV infected population,and analyzed the correlation between virus neutralization activity and viral load.1.The effect of HCV envelope protein mutation to HCV function.HCV envelope protein is a structural protein that is exposed to the surface of virus particles and plays an important role in virus infection and immune escape.In this study,after deep sequencing of the virus mutations which produced by the successive culture,it was found that there were F291V,T331S and T444P mutations on the envelope protein.F291V mutation was found in E1 protein,and T331S and T444P mutations were found in E2 protein.These mutations were introduced in the prototype plasmid,and the HCVcc containing these mutations was prepared.The E41/JFH1 strain containing F291V,T331S and T444P mutations,D42/JFH1 strains with T444P mutations and B43/JFH strains containing F291V mutations.And we hope to study the effects of envelope protein mutations on viral function by these HCVcc.The results showed that the mutations of envelope protein increased the titer of HCVcc by 10-15 times,and they did not affect the expression of virus E2 protein and the rate of virus invasion into host cells.But mutations not only improve the efficiency of virus packaging and release,but also increase the virus titer and the level of lipoprotein on the virus surface.At the same time the utilization of SR-BI receptor and lipoprotein signaling pathway was increased.In the absence of neutralizing antibody and the evolutionary pressure from the host immune system,the virus replication and packaging release efficiency is continuously improved,and the virus evolves in the direction of higher titer.It embodies the natural unity of the virus evolution in function and environment.2.The effect of envelope protein mutation on the neutralization of HCV antibody.In this study,the E41/JFH1 and J6/JFH1 strains HCVcc were neutralized by type 2a HCV serum.,it was found that the serum of HCV patients could significantly neutralize the infection of the E41/JFH1 mutant virus,but difficult to neutralize the J6/JFH1 virus infection.The APOE antibody could neutralize 76%of E41/JFH1 virus infection at 1:50 dilution,while only 55%of J6/JFH1 infection was neutralized.The neutralizing experiment was performed on E41/JFH1 and J6/JFH1 strains with the HCV envelope protein E2,77-661,3a,5A,and HVR1 antibody.The results showed that these antibodies were effective neutralize the infection of E41/JFH1 mutant viruses,70%,75%,and 65%,60%respectively.while the J6/JFH1 strain was difficult to neutralize.It indicated that these antibodies have cross-neutralization activity to the E41/JFH1 mutant virus,and the envelope protein mutations does not affect the neutralization epitopes of the virus.The high/low density viruses of E41/JFH1 and J6/JFH1 were neutralized by using 77-661,TW-SHC,APOE and CD81 antibodies.The results showed that the neutralization efficiency of 77-661 antibody to E41/JFH1 and J6/JFH1 high/low density virus was 76%,42%and 76%,42%respectively.which shows that 77-661 antibody can be more effective neutralize high density of the virus infection;And the neutralization efficiency of TW-SHC antibody to E41/JFH1 and J6/JFH1 high/low density virus was 65%,10%and 65%,10%respectively.Which indicates that the TW-SHC antibody is difficult to neutralize the high density J6/JFH1 virus,while the low density virus of E41/JFH1 is easy to neutralize.And the neutralization efficiency of APOE antibody to E41/JFH1 and J6/JFH1 high/low density virus was 45%,23%and 88%,37%respectively.which indicating that the envelope protein mutations increased the neutralization activity of the APOE antibody to the virus.While the CD81 antibody could efficiency neutralization of E41/JFH1 and J6/JFH1 high/low density virus,which indicating that mutations do not change the utilization of the virus to CD81 receptors,and there was no difference in the utilization of CD81 receptors by different density particles.The mutation of envelope protein changed the virus utilization of receptors and neutralizing activity,which reflects the unity of HCV on evolution and function.At the expense of the moderately reduce the infectivity,the virus avoid the neutralizing of antibody,but guarantee the persistent infection of virus,embody the characteristics of the virus to natural selection.At the same time,this study also identified amino acid residues in the HCV envelope protein are beneficial to viral immune escape,which is of great significance for the study of virus evolution and immune escape.3.A cross-sectional serum investigation of a clustering HCV infection in Southwest ChinaIn this part,the serum samples of HCV infection in a region of yunnan province were analyzed,and found that in the group which the HCV RNA level higher than 1 x 106 copy,the numerical of ALT and AST presented lower trend;in the analysis of HCV patient neutralization and E2 antibody level,we found that neutralization antibodies cannot effectively suppress viral replication in chronic HCV patients,which means neutralizing antibodies cannot protect HCV patients from chronic infection.This study explored the risk of HCV transmission among family members and analyzed the relationship between the liver function of the infected patients,the level of antibody and the viral load in the serum.It is proved that life contact do not transmit HCV,and the higher titer neutralization antibody of chronic HCV patients cannot play a protective role in patients.Summary:In this research,through studying the HCV envelope mutations,produced in the successive culture to the effects of virus infection characteristics,antibody neutralization and receptor utilization as well as serological characteristic clustering HCV infection,deepening the pathogenic mechanisms of HCV envelope protein and virus immune escape.