The Study On The Structure Of Hepatitis C Virus Core G-Guadruplex Structure And Its Protein Expresssion And Immunosuppression

Background:Hepatitis C virus(HCV)is a single-stranded positive strand RNA virus of flaviviruses family which mainly invades host liver cells.HCV genome is a multi-cistron and all genes about 10 Kb locate in its RNA chain.After HCV invasion to the host,the genome encoded as a polyether chain,and using the host and the virus itself enzyme cleaved the polypeptide into multiple structural proteins(Core,E1 and E2)and nonstructural proteins(p7,NS2～NS5B).HCV infection is mainly through blood-borne way,which can cause hepatitis,liver fibrosis and even Hepatocellular carcinoma(HCC).There is about 70 million people infection of HCV around the world,80%of HCV patients develop into chronic persistent infection.In natural conditions,the immunogenicity of HCV is very low,leading to the lack of HCV specific vaccine.There have been a variety of direct antiviral drugs(DAA)such as sofosbuvir,dasabuvir produced in developed countries,which have a good therapeutic effect on HCV infection.The pegylated interferon combined with ribavirin administration is the major treatment for HCV in clinicals in our country.However,there are a lot of disadvantages about this therapy,including the expensive price,less viral response,and some side effects.Therefore,it is necessary for developing the new HCV vaccine for prevention and treatment of HCV.Objective:Core is one of important structural proteins for HCV.Our recent work has demonstrated that the G-quadruplex(G4)structure existed on HCV core gene.This study will further study the structure of HCV core G4 structure and its protein expresssion and immunogenicity in vivo.Methods:We used circular dichroism(CD)to detect the G4 spatial structure of the G4 oligonucleotide chain RNA(named as G4R)and its site-specific mutant of G4(named as G4RM),comparison of the structural stability.The HCV wild-type core gene G4(DNA)sequence was mutated as G4M-core by PCR site-specific mutant.Then wild type and mutated core genes were constructed into the eukaryotic expression plasmid pcDNA3.1-Myc,which pcDNA3.1-core-G4-WT(named as pG4)and pcDNA3.1-core-G4-M(named as pG4M)transfected into Huh.7.5.1 cells,and the expression of core protein was examined by western blot.The mice were immunized with the pG4 and pG4M plasmids DNA respectively,and their humoral and cellular responses were examined.Results:The melting curve analysis of CD showed the melting temperature of G4RM was lower than that of G4R,which indicates that G4RM structure is unstable.Western blot analysis showed that pG4M had much higher Core protein expression level compared to pG4.Analysis of animal immunization showed that pG4M induced higher levels of total IgG and IFN-y compared to pG4 in serum.By enzyme-linked immunospot(ELISpot)assay,we found that the release IFN-y level of splenic CD4+T in the pG4M treated group was much higher than those of pG4 treated groups.Flow cytometry showed that the intracellular IFN-γ production in the splenic CD4 + T cells was much higher than those of pG4.The secretion of IL-12 from the splenic DC in the pG4M treated group was also much higher than those of pG4 treated group.Conclusion:In summary,those results demonstrate that G4 structure of HCV core can inhibit its protein translation.The mutation of core G4 structure increased Thl-type immune responses.G4 mutation of HCV core gene can enhance their own protein expression and lead to the instability of its structure.Our research provides a new strategy of exploring HCV DNA vaccine by enhancing immunogenicity of core with G4 mutant.