AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting ...AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.展开更多
基金Supported by The Natural Science Foundation of China,No. 30872247 and 30600529the PLA medical research funds of China,No. 06H021 and 06Z027 and Shanghai LAD Project (B901)
文摘AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.
