It is likely that hepatocyte debris generated secondary to intrinsic production of viral dsRNA in HCV-infected hepatocytes affect the antiviral effector response of the immune system through maturation of dendritic cells. Hence, the effector cell activation may be enhanced by the induction of cell death through the IPS-1 pathway in hepatocytes which may facilitate producing dsRNAcontaining debris. In comparison to the JFH1GND construct with deficient replication that showed a rapid reduction in its RNA levels over time after FPL 64176 transfection into mouse hepatocytes, J6JFH1 RNA was detected at SKF 97541 four-log higher levels and was maintained at a relatively stable levels in IPS-1ko hepatocytes. Although the number of mouse cells expressing HCV proteins was found to increase over time, as detected by IF, the ratio between HCVnegative and -positive cells did not show any significant change for 7 days after transfection and increased after 10 days. This indicates a negative selection of HCV-bearing cells over time which may be due to slower cellular replication, or loss of HCV replication. Another possibility may be that HCV infection is affected by the presence of an inhibitory factor possibly triggered by HCV replication or the lack of a human host factor required for HCV replication. Due to the initial replication of HCV in the transfected IPK and IRK mouse hepatocytes for the first 7 days and the establishment of infection, we favor the presence of a possible inhibitory factor that may be triggered by HCV replication. Another factor that also limits HCV spread in mouse hepatocytes is the failure of HCV to produce infectious particles in these cells. Using this newly established immortalized mouse hepatocyte line, we found that although J6JFH1, JFH1FL and the subgenomic JFH1 replicon all share a similar non-structural region derived from isolate JFH1 that is required for HCV replication, and although all of these constructs can replicate efficiently in HuH7.5.1 cells, strikingly, only J6JFH1 carrying the J6 structural region replicated in mouse hepatocytes. This indicates the importance of the J6 structural region and/or the chimeric construct between J6 and JFH1 for HCV replication in mouse hepatocytes. Structural regions are known to be important for HCV entry and/or particle formation, but this is the first time that their importance in replication in HCV-bearing cells has been demonstrated.