2A). HCV-infected IHHs displayed LC3-II expression, whereas a predominant LC3-I band was observed in HCV-infected siBCN1 IHHs (data not shown). Next, control IHHs and siBCN1 IHHs were infected with cell culture–grown HCV genotype 1a or 2a (H77 or JFH1 clone) for 3 days. We determined the effect of BCN1 knockdown on the release

of infectious virus particles. The HCV titer in BCN1-knockdown infected hepatocytes was reduced in comparison with HCV-infected control IHHs (Fig. 2B). Therefore, the impairment of autophagy machinery reduces HCV production in IHHs. We have seen earlier that HCV-infected IHHs display inhibition of IFN-α and IFI27 expression.23 Next, we asked whether the knockdown of the autophagy protein after HCV infection modulates the IFN signaling pathway. For this, the expression levels of IFN-β, OAS1, IFN-α, and IFI27 from control IHHs and siBCN1 IHHs were initially GPCR & G Protein inhibitor examined to verify that BCN1 knockdown has an effect on the IFN signaling pathway. A significant difference in the expression of these genes in control IHHs and siBCN1 IHHs was not observed (data not shown). We next examined the status of a number of IFN-stimulated genes in HCV-infected control IHHs and siBCN1 IHHs. We showed earlier that HCV infection

in IHHs up-regulates IFN-β and PI3K Inhibitor Library OAS1.24 Here, the mRNA level of IFN-β and OAS1 was 6- to 10-fold higher in siBCN1 IHHs infected with HCV genotype 1a or 2a versus virus-infected control IHHs (Fig. 3). Because HCV infection of IHHs inhibits IFN-α and IFI27 mRNA expression, we wanted to examine whether IFN-α synthesis is altered in HCV-infected siBCN1 IHHs. The results displayed a significant increase in IFN-α and its downstream molecule IFI27 expression in HCV-infected siBCN1 DNA ligase IHHs versus HCV-infected control IHHs (Fig. 3). Together, these results indicate that HCV-induced autophagy suppresses the expression of IFN-stimulated genes. To further confirm the modulation of IFN-stimulated genes in HCV-infected

autophagy-knockdown cells, we used another autophagy protein, ATG7. Knockdown of ATG7 in IHHs (siATG7 IHHs) did not alter the cell viability or display any off-target effect (data not shown). Control IHHs or siATG7 IHHs were infected with HCV (genotype 1a) for 72 hours. Virus release was measured, and a decrease in HCV growth in siATG7 IHHs versus HCV-infected control IHHs was observed (Fig. 4A). OAS1 (Fig. 4B), IFNA1 (Fig. 4C), and IFI27 mRNA expression (Fig. 4D) was up-regulated in HCV-infected siATG7 IHHs versus HCV-infected control IHHs (Fig. 4). Similar results were obtained when cells were infected with HCV genotype 2a. Together, these results suggest that disruption of autophagy machinery induces the IFN-signaling pathway in HCV-infected hepatocytes. We hypothesized that autophagy promotes the survival of HCV-infected cells for virus persistence.