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A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.

Ranjith-Kumar CT, Wen Y, Baxter N, Bhardwaj K, Cheng Kao C - PLoS ONE (2011)

Bottom Line: 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.The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling.The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America. ctrkumar@indiana.edu

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.

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A cell-based assay for the HCV 1b polymerase.A) Schematic for the 5BR assay. The third step in the protocol, identified in parenthesis, is designed to confirm that a treatment acted through the HCV polymerase rather than the RIG-I signaling pathway. It was left out in some assays. B) Results for the 5BR assay demonstrating that expression of the 1b HCV polymerase can induce RIG-I-dependent luciferase production in HEK 293T cells. Ratio in the vertical axis denotes the units of firefly luciferase driven from the interferon β promoter relative to the Renilla luciferase driven from the TK promoter. The cells were transfected to express the NS5B construct denoted below the horizontal axis along with either an empty pUNO vector (Vec. only) or pUNO-RIG-I (RIG-I). The white bars show the ratios of the two luciferases in the absence of exogenously provided ligands. The grey bars show the results from cells transfected with 3PdsR24, an agonist of RIG-I. The numbers above the bars show the mean of at least three independent trials and error bars show standard deviation. The RIG-I ligand, 3PdsR24, was transfected into cells at 50 nM final concentration and serves as a check for whether RIG-I is responsive to an agonist. C) Mutations in the RIG-I protein will abolish the output in the 5BR assay. The ratios of the firefly and Renilla luciferases with standard deviations in parentheses are shown. D) TLR3 co-expressed in HEK 293T cells did not respond to RNA synthesis by NS5B. Effects of 1b5B on RIG-I and TLR3 signaling in the presence and absence of agonists are shown in the graph. Where used, poly(I:C) (labeled as pIC in the graph) was added to the medium of cells to a final concentration of 1 µg/ml, and the RIG-I agonist 3PdsR24 was transfected at 50 nM. E) NS5B can also induce MDA5 to activate luciferase reporter production. pUNO-MDA5 was co-transfected into the HEK 293T cells along with the plasmids expressing the RNA synthesis competent or incompetent NS5Bs. Poly(I:C) was transfected into the cells to serve as an exogenously-provided agonist to MDA5.
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pone-0022575-g001: A cell-based assay for the HCV 1b polymerase.A) Schematic for the 5BR assay. The third step in the protocol, identified in parenthesis, is designed to confirm that a treatment acted through the HCV polymerase rather than the RIG-I signaling pathway. It was left out in some assays. B) Results for the 5BR assay demonstrating that expression of the 1b HCV polymerase can induce RIG-I-dependent luciferase production in HEK 293T cells. Ratio in the vertical axis denotes the units of firefly luciferase driven from the interferon β promoter relative to the Renilla luciferase driven from the TK promoter. The cells were transfected to express the NS5B construct denoted below the horizontal axis along with either an empty pUNO vector (Vec. only) or pUNO-RIG-I (RIG-I). The white bars show the ratios of the two luciferases in the absence of exogenously provided ligands. The grey bars show the results from cells transfected with 3PdsR24, an agonist of RIG-I. The numbers above the bars show the mean of at least three independent trials and error bars show standard deviation. The RIG-I ligand, 3PdsR24, was transfected into cells at 50 nM final concentration and serves as a check for whether RIG-I is responsive to an agonist. C) Mutations in the RIG-I protein will abolish the output in the 5BR assay. The ratios of the firefly and Renilla luciferases with standard deviations in parentheses are shown. D) TLR3 co-expressed in HEK 293T cells did not respond to RNA synthesis by NS5B. Effects of 1b5B on RIG-I and TLR3 signaling in the presence and absence of agonists are shown in the graph. Where used, poly(I:C) (labeled as pIC in the graph) was added to the medium of cells to a final concentration of 1 µg/ml, and the RIG-I agonist 3PdsR24 was transfected at 50 nM. E) NS5B can also induce MDA5 to activate luciferase reporter production. pUNO-MDA5 was co-transfected into the HEK 293T cells along with the plasmids expressing the RNA synthesis competent or incompetent NS5Bs. Poly(I:C) was transfected into the cells to serve as an exogenously-provided agonist to MDA5.

Mentions: We seek to use innate immune signaling to develop a cell-based assay to analyze RNA synthesis by the HCV polymerase. A typical assay used HEK 293T cells transfected with four plasmids to express RIG-I, NS5B, and two luciferases that report from the IFN-β promoter and a thymidine kinase promoter (Fig. 1A). The latter serves to control for transfection efficiency and cellular toxicity. The typical assay yielded a ca. four- to nine-fold increase in the IFN-β luciferase signal when the cells were transfected with 1b5B (NS5B of genotype 1b) and a six- to twelve-fold increase was observed with the 1bΔ21, which lacks the C-terminal transmembrane helix of 21 residues (1bΔ21) (Fig. 1B). 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.


A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.

Ranjith-Kumar CT, Wen Y, Baxter N, Bhardwaj K, Cheng Kao C - PLoS ONE (2011)

A cell-based assay for the HCV 1b polymerase.A) Schematic for the 5BR assay. The third step in the protocol, identified in parenthesis, is designed to confirm that a treatment acted through the HCV polymerase rather than the RIG-I signaling pathway. It was left out in some assays. B) Results for the 5BR assay demonstrating that expression of the 1b HCV polymerase can induce RIG-I-dependent luciferase production in HEK 293T cells. Ratio in the vertical axis denotes the units of firefly luciferase driven from the interferon β promoter relative to the Renilla luciferase driven from the TK promoter. The cells were transfected to express the NS5B construct denoted below the horizontal axis along with either an empty pUNO vector (Vec. only) or pUNO-RIG-I (RIG-I). The white bars show the ratios of the two luciferases in the absence of exogenously provided ligands. The grey bars show the results from cells transfected with 3PdsR24, an agonist of RIG-I. The numbers above the bars show the mean of at least three independent trials and error bars show standard deviation. The RIG-I ligand, 3PdsR24, was transfected into cells at 50 nM final concentration and serves as a check for whether RIG-I is responsive to an agonist. C) Mutations in the RIG-I protein will abolish the output in the 5BR assay. The ratios of the firefly and Renilla luciferases with standard deviations in parentheses are shown. D) TLR3 co-expressed in HEK 293T cells did not respond to RNA synthesis by NS5B. Effects of 1b5B on RIG-I and TLR3 signaling in the presence and absence of agonists are shown in the graph. Where used, poly(I:C) (labeled as pIC in the graph) was added to the medium of cells to a final concentration of 1 µg/ml, and the RIG-I agonist 3PdsR24 was transfected at 50 nM. E) NS5B can also induce MDA5 to activate luciferase reporter production. pUNO-MDA5 was co-transfected into the HEK 293T cells along with the plasmids expressing the RNA synthesis competent or incompetent NS5Bs. Poly(I:C) was transfected into the cells to serve as an exogenously-provided agonist to MDA5.
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Related In: Results  -  Collection

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pone-0022575-g001: A cell-based assay for the HCV 1b polymerase.A) Schematic for the 5BR assay. The third step in the protocol, identified in parenthesis, is designed to confirm that a treatment acted through the HCV polymerase rather than the RIG-I signaling pathway. It was left out in some assays. B) Results for the 5BR assay demonstrating that expression of the 1b HCV polymerase can induce RIG-I-dependent luciferase production in HEK 293T cells. Ratio in the vertical axis denotes the units of firefly luciferase driven from the interferon β promoter relative to the Renilla luciferase driven from the TK promoter. The cells were transfected to express the NS5B construct denoted below the horizontal axis along with either an empty pUNO vector (Vec. only) or pUNO-RIG-I (RIG-I). The white bars show the ratios of the two luciferases in the absence of exogenously provided ligands. The grey bars show the results from cells transfected with 3PdsR24, an agonist of RIG-I. The numbers above the bars show the mean of at least three independent trials and error bars show standard deviation. The RIG-I ligand, 3PdsR24, was transfected into cells at 50 nM final concentration and serves as a check for whether RIG-I is responsive to an agonist. C) Mutations in the RIG-I protein will abolish the output in the 5BR assay. The ratios of the firefly and Renilla luciferases with standard deviations in parentheses are shown. D) TLR3 co-expressed in HEK 293T cells did not respond to RNA synthesis by NS5B. Effects of 1b5B on RIG-I and TLR3 signaling in the presence and absence of agonists are shown in the graph. Where used, poly(I:C) (labeled as pIC in the graph) was added to the medium of cells to a final concentration of 1 µg/ml, and the RIG-I agonist 3PdsR24 was transfected at 50 nM. E) NS5B can also induce MDA5 to activate luciferase reporter production. pUNO-MDA5 was co-transfected into the HEK 293T cells along with the plasmids expressing the RNA synthesis competent or incompetent NS5Bs. Poly(I:C) was transfected into the cells to serve as an exogenously-provided agonist to MDA5.
Mentions: We seek to use innate immune signaling to develop a cell-based assay to analyze RNA synthesis by the HCV polymerase. A typical assay used HEK 293T cells transfected with four plasmids to express RIG-I, NS5B, and two luciferases that report from the IFN-β promoter and a thymidine kinase promoter (Fig. 1A). The latter serves to control for transfection efficiency and cellular toxicity. The typical assay yielded a ca. four- to nine-fold increase in the IFN-β luciferase signal when the cells were transfected with 1b5B (NS5B of genotype 1b) and a six- to twelve-fold increase was observed with the 1bΔ21, which lacks the C-terminal transmembrane helix of 21 residues (1bΔ21) (Fig. 1B). 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.

Bottom Line: 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.The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling.The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America. ctrkumar@indiana.edu

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.

Show MeSH
Related in: MedlinePlus