Original Article
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World J Gastroenterol. Dec 28, 2014; 20(48): 18284-18295
Published online Dec 28, 2014. doi: 10.3748/wjg.v20.i48.18284
Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes
Xin Cheng, Xiang-Cui Gao, Jun-Ping Wang, Xin-Ying Yang, Yan Wang, Bao-Sheng Li, Fu-Biao Kang, Hai-Jun Li, Yue-Min Nan, Dian-Xing Sun
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
Yue-Min Nan, Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang 050082, Hebei Province, China
Author contributions: Cheng X and Gao XC contributed equally to this work; Sun DX and Cheng X designed the research; Cheng X, Gao XC, Wang JP, Yang XY, Wang Y, Li BS, Kang FB and Li HJ performed the research; Sun DX, Cheng X, Gao XC and Nan YM analyzed the data; Sun DX and Cheng X wrote the paper.
Supported by Grants from the Study on Prevention and Control of Viral Hepatitis in the Key Program for Science and Technology Development of Hebei Province, No. 10276102D
Correspondence to: Dian-Xing Sun, MD, Professor of Medicine, The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, 398 West Zhongshan Road, Shijiazhuang 050082, Hebei Province, China. sundianxing@hotmail.com
Telephone: +86-311-87978434 Fax: +86-311-87978434
Received: January 29, 2014
Revised: August 28, 2014
Accepted: October 15, 2014
Published online: December 28, 2014
Processing time: 341 Days and 11.2 Hours
Abstract

AIM: 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 × 108± 2.747 × 107vs 5.368 × 107± 1.016 × 107, P < 0.05; 1.049 × 108± 2.747 × 107vs 5.243 × 107± 1.194 × 107, 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.

CONCLUSION: Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

Keywords: Hepacivirus; Replicon; Internal ribosome entry site; Tricistronic expression

Core tip: Two sequential RNA-binding motif protein 3 internal ribosome entry sites (IRESs) of 22 nucleotides were used to construct a tricistronic hepatitis C virus (HCV) replicon, initiating translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating translation of neomycin resistance gene. Intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in cells transiently and stably transfected with tricistronic replicon RNA were comparable to those in cells transfected with traditional bicistronic HCV replicons. The novel replicon could be applied to evaluate replication inhibition efficacy of anti-HCV agents, except for interferon-α2b, which might be attributed to suppressed interferon response pathway.