Study On The Functions Of Different Truncated Core Proteins Of Genotype 1b Hepatitis C Virus

Core protein (CORE)-the nucleocapsid of hepatitis C virus (HCV) exhibits an important role in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC). The structures and functions of CORE among different HCV genotypes have some differences and different domains of CORE contain different functions. Genotype 1b is the prevalent genotypes all over our country. Hence, it has important significances to conduct researches on the roles of different truncated CORE of genotype 1 b in the pathogenesis of HCV persistent infection and HCC.Protein kinase R (PKR) has been taken into especially great consideration recently. Since it is a key molecule not only for anti-virus but also the establishment of HCV persistence and HCC. Signal transducer and activator of transcription (STAT) family proteins are transcription factors critical in mediating cellular signal. Among them, STAT3 is often constitutively phosphorylated (p-STAT3) in human cancers and implicated in tumor-genesis. Constitutive activation of STATS has been demonstrated to be associated with malignant transformation induced by various onco-proteins. Based on the findings that CORE derived from HCC could directly bind to PKR and CORE could also activate STAT3 and induce cell transformation. PKR could also act as an adaptor. In this study, we conducted research on the binding among CORE, PKR and STAT3, identifying the domains related to their interactions, and hypothesizing the models among them.Objective(1) To investigate the roles of different truncated genotype lb CORE in the pathogenesis of-HCV persistent and HCC.(2) To study if HCV or CORE could influence the expression of PKR and STAT3.(3) To investigate the role of interactions of CORE, PKR and STAT3 in the pathogenesis of HCV and HCC.(4) To identify the domains related to their interactions and hypothesize the model of interactions among CORE, PKR and STAT3.Methods(1) Seven different truncated glutathione-S-transferase (GST)-Core fusion plasmids were constructed, expressed and purified. Four different truncated GST-PKR fusion proteins were expressed and purified.(2) Expression levels of PKR, STAT3, p-STAT3 among different cell lines were assayed.(3) Three different methods including co-immunoprecipitation, con-focal analysis, GST pull-down and western blot were used to see if CORE, PKR and STAT3 could bind with one another and map the domains related to their interactions.Results(1) Different truncated genotype lb CORE were successfully constructed and expressed.(2) Different truncated GST-CORE and GST-PKR fusion proteins were expressed with different quantities. Except the fragment of 59-126 amino acid (aa), the longer the fragment, the less expressed. CORE dimmer depended on 59-126 aa. Full-length of GST-PKR expressed than other truncated fragment.(3) The direct binding between CORE and PKR depended on CORE N-terminus 1-58 aa and PKR N-terminus 1-180 aa. N-terminus 1-126 aa contributed to CORE and STAT3 interaction. PKR binds to STAT3 depended on the full-length of PKR.(4) Different HCV strains and different truncated CORE bind to STAT3 and p-STAT3 with different intensities. HCV could induce the expression of STAT3 and increase the activity of PKR.Conclusions(1) Successful construction of different truncated genotype lb CORE expression plasmids paid the basis for the further study of HCV CORE structure, antigen, molecular immunity and biological characteristic.(2) These findings suggest that CORE, PKR and STAT3 can interact with each other to form trimer. CORE plays its function through both of PKR and STAT3; or HCV CORE binding to STAT3 and activating STAT3 might be due to Core/ PKR interaction.(3) CORE interacts directly with PKR. The distinct interaction among CORE, PKR and STAT3 might provide a new molecular mechanism of HCV pathogenesis and play an important role in the pathogenesis of HCV persistent infection and HCC.