title: [Construction and sequence analysis of recombinant HCV-1b replicon by replacing NS5A region].
abstract: OBJECTIVE
  To construct the recombinant HCV-1b replicon by replacing NS5A region using serum samples from patients with chronic hepatitis C (CHC) in South China and explore the biological characteristics of NS5A protein in response to antiviral therapy.
  
  
  METHODS
  The on-off plasmid containing the cutting sites of the restriction endonucleases MIu I and Bcl I was designed based on the backbone of robust HCV 1b replicon. The full-length fragments of HCV NS5A were amplified from different CHC patients by RT-PCR and cloned into pMD-18 vector, followed by sequence analysis of amino acid mutation of ISDR, PKRBD, V3 and IRRDR within the NS5A region. If the amplicon obtained contained no MIu I or Bcl I cutting sites, the NS5A fragment was re-amplified using primers containing the cutting sites and inserted into the replicon for replacement.
  
  
  RESULTS
  The full-length fragments of NS5A were obtained successfully from CHC patients. The core region of ISDR-V3 of NS5A was replaced in the HCV replicon plasmid and showed correct sequences. The amino acid mutations of ISDR and PKRBD within NS5A were more frequent in patients with sustained viral response (SVR) than those without SVR. A high variability in the amino acid sequence was observed in both IRRDR and V3 regions.
  
  
  CONCLUSIONS
  The plug-in type recombinant HCV replicon for replacement of NS5A region in the virus from CHC patients has been successfully constructed, which provides a basis for further investigation of the biological characteristics of NS5A protein, the mechanisms of interferon-resistance, and antiviral therapy of difficult-to-treat CHC.
authors.affiliation: Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China. lijingtao555@163.com
authors.forename: Jingtao
authors.initials: J
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Yuanping
authors.initials: Y
authors.lastname: Zhou
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zeng
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Junjie
authors.initials: J
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Shuwen
authors.initials: S
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000008:administration & dosage / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D007372:Interferons / Q000008:administration & dosage / Q000494:pharmacology; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012093:Replicon / Q000235:genetics*; D017421:Sequence Analysis; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D007372:Interferons; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase