Abstract
RNA synthesis by the genotype 1b hepatitis C virus (HCV) polymerase (NS5B) transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon β promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C-terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non-nucleoside inhibitor benzothiadiazines (BTDs) that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis.
Highlights
Hepatitis C virus (HCV) infects approximately 175 million people worldwide
A typical assay used HEK 293T cells transfected with four plasmids to express Retinoic acid-inducible gene I (RIG-I), nonstructural protein 5B (NS5B), and two luciferases that report from the interferon b (IFN-b) promoter and a thymidine kinase promoter (Fig. 1A)
When cells expressing RIG-I and 1b5B were further transfected with the RIG-I agonist 3PdsR24, an additional increase in luciferase activity was observed (Fig. 1B), indicating that the activity induced by NS5B had not reached saturation under these conditions
Summary
Hepatitis C virus (HCV) infects approximately 175 million people worldwide. 50% percent of the HCVinfected individuals will develop hepatocellular carcinoma or liver cirrhosis after chronic infection [1]. Current treatment for HCV uses pegylated interferon and ribavirin, but efficacy is limited and tolerance of the treatment is a major concern, in part due to genetic predisposition [2,3,4]. HCV is a single-stranded RNA virus that belongs to the Flaviviridae family. The HCV genomic RNA is 9.6 kb in length and encodes a polypeptide, which is processed by cellular and virallyencoded proteases to generate ten structural and nonstructural proteins. The nonstructural protein 5B (NS5B) is the RNAdependent RNA polymerase (RdRp), the catalytic subunit of the replicase complex. Based on the paradigm established with HIV/ AIDS and herpesvirus, NS5B is an important target for antiviral therapy
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