HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus classified in the Flaviviridae family. The single-strand, positive-sense viral RNA genome encodes a single polyprotein precursor that is processed into three structural proteins (C, E1, E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by host and viral proteases. The RNA genome of HCV has significant heterogeneity with six major genotypes and numerous subtypes. HCV infection is currently treated with alpha interferon (IFN)-based therapy. Although the treatment outcome is variable among the six major HCV genotypes, only 50% of treated patients infected with genotype 1 respond to therapy. The ability of HCV to establish persistent infection with great success in human has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses. Epidemiological studies suggest that up to 20% of acutely infected HCV patients can resolve the infection without treatment, which implies that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. The molecular mechanisms that regulate innate intracellular antiviral responses may therefore serve as pivotal points of control, potentially limiting host permissiveness for HCV replication and favorably modulating subsequent adaptive immune responses. A major consequence of virus infection is the induction of the type I interferons (IFN) including IFN/, which are cytokines of immune system with essential function in innate and adaptive immune response. Secreted IFN then triggers hundreds of IFN stimulated genes (ISGs) expression which establishes an antiviral state in the surrounding of virus-infected cells to limit viral replication, spread and infection. Double-stranded RNA (dsRNA) is the replication intermediate of RNA virus and is produced during viral replication. It has long been thought that dsRNA is a marker of virus infection because its molecular characteristic seems to allow for the distinction of self and nonself RNA. Poly(I:C), which is synthetic dsRNA analogue, has been used widely as an alternative to viral dsRNA for studying innate immunity mechanism in the cells because of its capability of activation of IFN. IFN/ production is usually measured by using ELISA or RT-PCR methods. The disadvantage of these methods is either time-consuming or insensitive. We have developed a novel reporter assay, IFN-SEAP, which secreted alkaline phosphatase (SEAP) is under the control of human IFN promoter sequence. It is designed for monitoring human IFN promoter activation by detection of SEAP activity in the culture media. We have demonstrated that SEAP activity in the media of cell culture is specifically induced by poly(I:C). This assay is about 40 to 150-fold more sensitive than IFN-Luc reporter assay in the cells we tested. Thus, such reporter assay will be very useful for studying innate immune responses to various stimuli because of its high sensitivity and convenience to time-course analysis. Using the IFN-SEAP reporter assay, we have characterized several genes which exert profound effect on activation of IFN gene in host after stimulation with HCV RNA or poly (I:C). Also, we have successfully identified and characterized several cell lines with specific knock-down genes related to innate immunity. Currently, we start to assess the infectivity of HCV patient serum samples on innate immunity knock-down cell lines. In the past year, Dr. Zhang in our group spent most of his efforts to elucidate the role of protein kinase R (PKR) in regulating HCV replication in cultured human hepatoma cells. As a sensor of double-stranded RNA, PKR is activated by autophosphorylation of PKR through binding of double-stranded RNA or 5-triphosphate RNA. The eukaryotic translation initiation factor2 (eIF2a) is the substrate of PKR. Activated PKR phosphorylates eIF2a at serine 51 and blocks the function of eIF2a, which results in the suppression of host cell translation initiation and protein synthesis in infected cells. In addition, activation of PKR is also required for the up-regulation of NF-kB expression. As a central transcription factor, NF-kB has a pivotal role in the regulation of immune response. It not only participates in the transcription of interferon beta, but also promotes the expression of hundreds of interferon stimulated genes. Recent studies has found that SOCS1 protein is employed to evade immune response in viral infection by inhibition of NF-kB activity. SOCS1, functions as an ubiquitin ligase, has been demonstrated to suppress NF-kB activity through degradation of p65. In another way, SOCS1 can reduce interferon beta production to downregualte TNF-alpha production, which results in an impaired antiviral effect. In addition, PKR involves in the regulation of endoplasmic reticulum stress (ER stress) through eIFalpha/ATF4/CHOP pathway. Endoplasmic reticulum is an important cellular organelle responsible for the folding and sorting of proteins. Accumulated data has suggested that ER is a major HCV assembly and process factory. These studies indicate that PKR may play an important role in the regulation of immune response to HCV replication. In our study, we utilized PKR sh/siRNA to investigate the molecular mechanism of PKR in regulation of innate immunity and the impact of knock-down PRK on the replication of HCV1a. Either by transient or stable knock-down of PKR, inhibition of PKR expression will enhance HCV replication in Huh-7.5.1 cells. Furthermore, the ablation of PKR expression will suppress the IFN beta response to the double-stranded RNA stimulatioon and HCV1a infection. In addition, we have also found that pharmacological inhibitors of PKR significantly enhance HCV1a replication in Huh-7.5.1 cells. In contrast, PKR inhibitors have been found to suppress HCV genotype 2a replication during in vitro culture system by other investigators. It has been suggested that use of PKR inhibitors, in conjunction with IFN/ribavirin, would be beneficial for the treatment of chronically HCV-infected patients. Our results however point to the opposite direction. Our group has also investigated the effect of several NF-kB inhibitors on persistent HCV1a replication in Huh-7.5.1 cells, and found one compound showing significant enhancement of HCV1a replication. This compound has been extensively discussed in the literatures about its anti-cancer activity. Since HCV is the leading cause of hepatocellular carcinoma, the use of this compound on HCV patients should be very closely monitored.