title: A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.
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.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America. ctrkumar@indiana.edu
authors.forename: C T
authors.initials: CT
authors.lastname: Ranjith-Kumar
authors.forename: Yahong
authors.initials: Y
authors.lastname: Wen
authors.forename: Nielson
authors.initials: N
authors.lastname: Baxter
authors.forename: Kanchan
authors.initials: K
authors.lastname: Bhardwaj
authors.forename: C
authors.initials: C
authors.lastname: Cheng Kao
mesh_terms: D001483:Base Sequence; D001581:Benzothiadiazines / Q000494:pharmacology; D055162:Biocatalysis; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D005838:Genotype; D057809:HEK293 Cells; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072640:Interferon-Induced Helicase, IFIH1; D016899:Interferon-beta / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic; D015398:Signal Transduction / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001581:Benzothiadiazines; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D011971:Receptors, Immunologic; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta; D008156:Luciferases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C452820:RIGI protein, human; C452815:IFIH1 protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1