1. Volume 117, Issue 5, Pages 1187-1197 (November 1999)
Hepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response 
Morris Paterson, Carl D. Laxton, Howard C. Thomas, Andrew M. Ackrill, Graham R. Foster 
Gastroenterology 
Volume 117, Issue 5, Pages (November 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Inhibition of antiviral response by NS5A. (A and B) Effect of NS5A in HT1080 cells. (A) Detection of NS5A expression. HT1080 cells transfected with NS5A (cell pool) were treated with 1 μmol/L muristerone A for 24 hours. Cells were harvested, and a Western blot was performed with a rabbit anti-NS5A serum along with an alkaline phosphatase–conjugated secondary antibody. Arrow indicates an NS5A band. (B) Antiviral assay with EMC virus. HT1080 cells transfected with NS5A (cell pool) were treated with 1 μmol/L muristerone A before addition of IFN-α. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent) before addition of IFN-α. Cells were infected with EMC virus, and cell viability was measured after 16 hours by staining with crystal violet and measuring the optical density of 570 nm. Triplicate wells were used; average data were plotted including the SEM. (C and D) Effect of NS5BGDD→PDD in HT1080 cells. (C) Detection of NS5BGDD→PDD expression. HT1080 cells transfected with NS5BGDD→PDD were grown and treated with muristerone A for 24 hours, and a Western blot was performed with a rabbit antiserum raised against NS5B. Arrow indicates an NS5B band. (D) Antiviral assay with EMC virus. HT1080 cells transfected with NS5B(GDD→PDD) were treated with 30 μmol/L muristerone A or untreated, and an antiviral assay was performed. (E) Antiviral assay with EMC virus in untransfected cells. Native HT1080 cells were treated with 1 μmol/L muristerone A for 24 hours, and an antiviral assay was performed.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
NS5A inhibition of IFN-α is dose dependent. (A) Detection of NS5A expression. HT1080 cells transfected with NS5A (cell pool) were treated with a titration of muristerone A for 24 hours. Cells were harvested, and a Western blot was performed. Arrow indicates an NS5A band. (B) Antiviral assay with EMC virus. HT1080 cells transfected with NS5A (cell pool) were treated with 1.11, 3.33, and 10 μmol/L muristerone A for 24 hours before the addition of an IFN-α titration. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent) before addition of IFN-α; 24 hours later, cells were infected with EMC virus and an antiviral assay was performed. Duplicate wells were used, and average data were plotted. (C) Inhibition and NS5A expression. Inhibition of the antiviral effects of IFN-α, as measured by Δlog (EC50), was plotted against muristerone A concentration. (D) Antiviral assay with EMC virus in cells expressing NS5BGDD→PDD. HT1080 cells transfected with a null mutant of NS5B (GDD → PDD) were treated with 3.33, 10, and 30 μmol/L muristerone A (to induce protein expression) for 24 hours before addition of an IFN-α titration.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Comparison of the inhibitory effects of NS5A-NR and NS5A-CR. (A–D) NS5A-NR and NS5A-CR expression in HT1080 cells. (A and B) Treatment with muristerone A titration. HT1080 cells transfected with NS5A-NR (cell pool) and NS5A-CR (cell pool) were grown and treated with a titration of muristerone A for 24 hours. Cells were harvested, and a Western blot was performed. Arrow indicates an NS5A band. (C and D) Equalization of expression levels. Growing HT1080 cells transfected with NS5A-NR and NS5A-CR were treated with 30 and 0.4 μmol/L muristerone A, respectively, for 24 hours. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent). Cells were harvested, and a Western blot was performed. Pooled rabbit antiserum (raised to NS5A) primary antibody was used in blot C, and mouse monoclonal anti-NS5 antibody from a commercial source in blot D. Arrow indicates an NS5A band. (E and F) Antiviral assay in HT1080 cells with EMC virus. Cells transfected with (E) NS5A-NR and (F) NS5A-CR were treated with 30 and 0.4 μmol/L muristerone A, respectively, for 24 hours before addition of an IFN-α titration. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent) before addition of IFN-α; 24 hours later, cells were infected with EMC virus and an antiviral assay was performed. Triplicate wells were used and average data plotted including the SEM. For HT1080 cells expressing NS5A-NR, Δlog (EC50) = −0.09; for HT1080 cells expressing NS5A-CR, Δlog (EC50) = 0.30.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Changes in the interferon sensitivity of cell lines expressing different NS5A proteins. The Δlog (EC50) values for the different cell lines are shown with the 95% confidence intervals. The NS5A protein derived from a patient who did not respond to IFN-α (NS5A-NR) had very little effect on the cellular response to IFN-α, whereas the protein derived from a patient who did respond to IFN-α inhibited the response. Cells expressing various mutated forms of NS5A (NS5A-RES and NS5A-SEN) are also shown, and the difference between these 2 clones was very slight and did not reach the level of statistical significance.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Comparison of the inhibitory effects of NS5A-RES and NS5A-SEN. (A–D) NS5A-RES and NS5A-SEN expression in HT1080 cells. (A and B) Treatment with muristerone A titration. HT1080 cells transfected with NS5A-RES and NS5A-SEN were grown and treated with a titration of muristerone A for 24 hours. Cells were harvested, and a Western blot was performed. Arrow indicates an NS5A band. (C and D) Equalization of expression levels. HT1080 cells transfected with NS5A-RES and NS5A-SEN were treated with 0.4 and 10 μmol/L muristerone A, respectively, for 24 hours. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent). Cells were harvested and Western blots performed. In C, pooled rabbit anti-serum (raised to NS5A) primary antibody was used; in D, mouse monoclonal anti-NS5 antibody from a commercial source was used. Arrows indicate an NS5A band. (E and F) Antiviral assay in HT1080 cells with EMC virus. HT1080 cells transfected with (E) NS5A-SEN and (F) NS5A-RES were treated with 0.4 and 10 μmol/L muristerone A, respectively, for 24 hours before addition of an IFN-α titration. Negative control cells (expression not induced) were treated with a similar quantity of ethanol (muristerone A solvent) before addition of IFN-α; 24 hours later, cells were infected with EMC virus and an antiviral assay was performed. Triplicate wells were used and average data plotted including the SEM. For HT1080 cells expressing NS5A-SEN, Δlog (EC50) = 0.25; for HT1080 cells expressing NS5A-RES, Δlog (EC50) = 0.41.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
6–16 mRNA induction. Ribonuclease protection assay investigating induction of 6–16 mRNA in response to IFN-α and dsRNA in transfected HT1080 cell pools expressing NS5A-NR or NS5A-CR. HT1080 cell pools transfected with NS5A-NR and NS5A-CR were grown and pretreated with 1 μmol/L muristerone A for 24 hours. IFN-α (16 hours) and polyI · polyC (4 hours) were added, and the levels of 6–16 mRNA induction were measured by ribonuclease protection using a 32P-labeled 6–16 complementary RNA probe (protected fragment, 190 base pairs). The results were normalized against a 127–base pair protected actin fragment. Two different concentrations of IFN-α were used: (A) high-level IFN-α (1000 IU/mL) and (B) low-level IFN-α (50 IU/mL).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Comparison of NS5A-NR and NS5A-CR amino acid sequences. The amino acid sequences of NS5A-NR and NS5A-CR were compared using GAP (GCG, Wisconsin Package). Amino acid differences are highlighted by open circles. Vertical bars indicate residue identity, and double dots and single dots represent large and small residue similarity, respectively. Dash represents an amino acid deletion. Serine residues 2197, 2201, and 2204 (polyprotein numbering) necessary for hyperphosphorylation,8 the ISDR,14 the PKR-binding domain,23 the potential nuclear localization signal (NLS), and V3 variable stretch are indicated. The amino acid sequences of NS5A-NR and NS5A-CR have been submitted to EMBL (accession numbers AJ and AJ133097, respectively).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions