title: Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution.
abstract: OBJECTIVE
  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 × 10(6) vs 4.27 ± 1.72 × 10(4), P = 0.032). The maximal level of HCV RNA in chimera was 5.6 ± 1.8 × 10(4) 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 × 10(5) vs 0.56 ± 0.90 × 10(5), 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 × 10(2) ffu/mL to 2.5 ± 1.4 × 10(4) ffu/mL.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cardiology, NO. 254 Hospital PLA, Tianjin 300142, China.
authors.forename: Yong-Zhi
authors.initials: YZ
authors.lastname: Wang
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Wang
authors.forename: Ming-Mei
authors.initials: MM
authors.lastname: Cao
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.forename: Lan-Juan
authors.initials: LJ
authors.lastname: Zhao
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
authors.forename: Hao
authors.initials: H
authors.lastname: Ren
authors.forename: Zhong-Tian
authors.initials: ZT
authors.lastname: Qi
mesh_terms: D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase