Show simple item record Kao, C. Cheng Bhardwaj, Kanchan Baxter, Nielson Wen, Yahong Ranjith-Kumar, C. T. 2012-04-04T17:01:57Z 2012-04-04T17:01:57Z 2011-07
dc.identifier.citation Ranjith-Kumar C.T., Y. Wen, N. Baxter, K. Bhardwaj, and C. Cheng Kao. 2011.A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A. PLoS ONE 6(7): e22575. en
dc.description.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 b 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. en
dc.description.sponsorship This work was funded by the National Institute of Allergy and Infectious Diseases grant 1RO1AI073335 and RA0750150 to CCK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. en
dc.language.iso en_US en
dc.publisher PLoS en
dc.relation.isversionof doi:10.1371/journal.pone.0022575 en
dc.rights Copyright:Ranjith-Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. en
dc.title A Cell-Based Assay for RNA Synthesis by the HCV Polymerase Reveals New Insights on Mechanism of Polymerase Inhibitors and Modulation by NS5A en
dc.type Article en

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