title: Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes.
abstract: OBJECTIVE
  To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.
  
  
  METHODS
  The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.
  
  
  RESULTS
  The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.
  
  
  CONCLUSIONS
  Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Xiang-Cui
authors.initials: XC
authors.lastname: Gao
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Jun-Ping
authors.initials: JP
authors.lastname: Wang
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Xin-Ying
authors.initials: XY
authors.lastname: Yang
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Bao-Sheng
authors.initials: BS
authors.lastname: Li
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Fu-Biao
authors.initials: FB
authors.lastname: Kang
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Li
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Yue-Min
authors.initials: YM
authors.lastname: Nan
authors.affiliation: Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Dian-Xing Sun, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China.
authors.forename: Dian-Xing
authors.initials: DX
authors.lastname: Sun
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter; D016679:Genome, Viral; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D020871:RNA Stability; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Hepacivirus; Internal ribosome entry site; Replicon; Tricistronic expression