Objective To investigate the effect of G-quadruplex (G4) RNA structure of core of hepatitis C virus (HCV) on the specific immune response. Methods Circular dichroism (CD) was usedto detect the G4 spatial structure of the G4 oligonucleotide chain RNA (named as G4R) and its mutant of G4 (named as G4RM) by G base site-specific mutation.The HCV wild-type core gene G4(DNA) sequence was mutated as G4M-core by PCR site-directed mutagenesis without changing the amino acid codon.Then wild type and mutated core genes were constructed into the eukaryotic expression plasmid pcDNA3.1-Myc, and produced as pcDNA3.1-core-G4-WT (named as pG4) and pcDNA3.1-core-G4-M (named as pG4M), 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 CD results showed that the structure of G4RM was changed compared to Wild type G4R, and the melting curve analysis showed the melting temperature of GR4M was lower than that of G4R, which indicates that G4RM structure is unstable. Western blot analysis showed that pG4M had much higher protein expression level compared to pG4(P<0.05). Analysis of animal immunization showed that pG4M induced increased levels of total IgG and IFN-γ compared to pG4(P<0.05). The IgG level of the pG4M group was 1.61 times higher than that of the pG4 group. By enzyme-linked immunospot(ELISpot)assay, we found that the release IFN-γ level of pG4M was 1.39 times higher than those of pG4. Flow cytometry showed that the intracellular IFN-γ production in the splenic CD4+ T cells was 1.79 times than those of pG4. Conclusion The G-quadruplex structure of HCV core can inhibit its protein translation. The mutation of G-quadruplex of core led to increased Th1-type immune responses. This is the first report demonstrate that HCV core G-quadruplex mutation can enhance its immunogenicity and could be used as a new strategy ofexploring HCV vaccine with enhanced immunogenicity.

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