Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes

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 andsuccessive antiviral screening using a luciferase marker.Briefly,two sequential Rbm3 IRESes were inserted into bicistronic p UC19-HCV plasmid,consequently forming a tricistronic HCV replicon(p HCV-rep-Neo R-h Rluc),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 s H7 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 s H7 cells and cells transiently transfected with tricistronic replicon RNA.RESULTS:The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV nonstructural gene in s H7 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 p HCV-rep-Neo R-h Rluc group was approximately 2-fold of those in the p UC19-HCV-h RLuc and Tri-JFH1 groups(1.049×108±2.747×107 vs 5.368×107±1.016×107,P<0.05;1.049×108±2.747×107 vs 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 p HCV-rep-Neo R-h Rluc and p UC19-HCV-h RLuc groups were similar(159.619±9.083 vs163.536±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 s H7 cells.After interferon stimulation,Mx A m RNA and protein levels were lower in s H7 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.