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. 2001 Jul;75(13):6095-106.
doi: 10.1128/JVI.75.13.6095-6106.2001.

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response

S J Polyak  1 , K S Khabar, D M Paschal, H J Ezelle, G Duverlie, G N Barber, D E Levy, N Mukaida, D R Gretch
Affiliations

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response

S J Polyak et al. J Virol. 2001 Jul.

Abstract

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

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Figures

FIG. 1
FIG. 1
Effect of NS5A expression on IFN signal transduction. (A) Tetracycline (Tc)-regulated expression of full-length and mutant NS5A proteins. FL-NR denotes a full-length NS5A construct derived from an IFN-nonresponsive patient, while FL-CR denotes a full-length NS5A construct derived from an IFN-responsive patient. ΔN-110, ΔN-222, ΔN-334, ΔC-117, and ΔC-230 represent NS5A deletion mutants lacking 110, 222, 334, amino acids from the amino terminus and 117 and 230 amino acids from the carboxy terminus, respectively. Protein positions are shown by arrows. Western blots were probed with HCV-infected patient serum as described in Materials and Methods. (B) Effect of NS5A expression on ISGF-3 induction. Plasmids were transiently transfected into HeLa Tet-Off cells, grown in the absence and presence of tetracycline to induce and repress NS5A, respectively, and treated with IFN to induce ISGF-3. Protein extracts were hybridized to a 32P-labeled oligonucleotide corresponding to the consensus ISRE. “puc” represents a control transfection with the pTRE-puc parental plasmid with no insert. “E3L,” “PKR,” and “E1A” represent expression of the E3L, PKR, and E1A proteins, respectively. The p48 monoclonal antibody used to form supershifts (denoted by “ss” and an arrow) is denoted by “p48 Mab”. “n” and “c” represent nuclear and cytoplasmic extracts, respectively. ISGF-3 is induced only with IFN treatment and is indicated with arrows.
FIG. 2
FIG. 2
Effect of NS5A expression on IFN-induced posttranslational modifications and upregulation of gene expression. (A) Effect of NS5A expression on IFN induction of STAT-1 phosphorylation and PKR expression. FL-NS5A-NR and FL-NS5A-CR proteins were repressed or induced by growing cells in the presence or absence of doxycycline (Dox) for 24 h and treated with IFN, and protein extracts were harvested at various times thereafter. Protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, followed by Western blotting with antibodies to NS5A (first panels), phosphorylated STAT-1 (second panels), PKR (third panels), and β-tubulin (fourth panels) as described in Materials and Methods. (B) Effect of NS5A expression on IFN-induced upregulation of MHC antigen. HeLa cells expressing FL-NS5A-NR (left panels) and FL-NS5A-CR (right panels) proteins for 24 h were treated with IFN, and cells were prepared for flow cytometry as described in Materials and Methods. Top panels are from cells grown in the presence of doxcycline (NS5A expression repressed), while bottom panels are from cells grown in the absence of doxcycline (NS5A expression induced). The gray histogram on the left represents a control staining with an isotype-matched control. To the right of the control histogram in each panel are the specific staining patterns. IFN-treated cells are depicted by the gray curve, while cells not treated with IFN are depicted by the black curve.
FIG. 3
FIG. 3
NS5A expression increases the expression of IL-8 mRNA. (A) Tetracycline-regulated expression of NS5A in human cells. HeLa cells expressing FL-NS5A-NR were incubated in medium with or without tetracycline for 48 h to repress or induce NS5A expression, respectively. Cells were then treated or not treated with IFN (20 U/ml) for 24 h. Cell extracts were prepared, and equal amounts of total cellular proteins were analyzed by Western blotting. The positions of the 58-kDa NS5A and positive control, NS5-(superoxide dismutase [SOD]), proteins are shown with arrows. (B) Semiquantitative detection of IL-8 mRNA by RT-PCR. cDNA was prepared from HeLa cells expressing or not expressing FL-NS5A-NR and treated or not treated with IFN. Serial dilutions of 1:2, 1:4, 1:8, 1:24, and 1:96 of cDNA (depicted above each lane with 2, 4, 8, 24, and 96, respectively) were subjected to PCR using IL-8-specific primers. Positive and negative controls (ctrl) are depicted with + and − signs, respectively. Amplification of cDNA generated without reverse transcriptase produced no PCR signal, indicating that the observed IL-8 PCR products were derived from IL-8 mRNA. Lanes M, molecular size markers. (C) Detection of IL-8 mRNA by RNase protection assay. Equal amounts of total cellular RNA from HeLa cells expressing or not expressing FL-NS5A-NR were hybridized to a 32P-labeled riboprobe cocktail containing probes for various human chemokines, including IL-8. Hybrids resistant to RNase digestion were separated on denaturing acrylamide gels, and autoradiography was performed. The positions of IL-8, control L32, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs are indicated with arrows. Note that the size of the protected fragment is smaller than the probe size because the probe contains vector-derived sequences that do not hybridize with target mRNA.
FIG. 4
FIG. 4
NS5A expression is associated with increased IL-8 protein production and inhibition of the antiviral effects of IFN. (A) Correlation among levels of IL-8 in culture supernatants, amounts of NS5A protein expression, and levels of EMCV rescue in the trans rescue assay. HeLa cells expressing FL-NS5A-NR were grown for 48 h in medium containing 0, 0.001, 0.01, and 1.0 μg of tetracycline per ml for 48 h, treated with 20 U of IFN per ml for 24 h, and infected with EMCV at an MOI of 0.01 for 24 h. The amount of IL-8 protein in culture supernatants, determined by ELISA, is indicated in the bar graph. Changes in EMCV titers were determined 24 h postinfection by viral plaque assay. The levels of NS5A protein expression were determined by Western blot analysis of equal amounts of total cellular protein extracts and are presented as +++, ++, +, and −, which correspond to high, intermediate, low, and undetectable levels of NS5A protein, respectively, as determined by computerized scanning of the chemiluminescent signal. Fold EMCV rescue represents the difference in EMCV titers in the presence of IFN among cells treated with 0, 0.001, and 0.01 μg of tetracycline per ml versus the 1.0-μg/ml concentration. (B) IL-8 inhibits the antiviral actions of IFN in vitro. HeLa Tet-Off cells were pretreated with or without 33 ng of recombinant human IL-8 per ml for 6 h and then with 20 U of IFN per ml for 18 h. Cells were then infected with EMCV at an MOI of 0.1 for 24 h. Supernatants were harvested, and the amount of EMCV was determined by titration on L929 cells. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.
FIG. 5
FIG. 5
NS5A transactivates the IL-8 promoter. (A) Effect of NS5A expression on luciferase expression under the control of the 546-luc IL-8 promoter. The 546-luc plasmid and pTRE-FL-NS5A-NR (10 μg each) were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated or not treated with IFN for 24 h. The amount of luciferase activity in cell lysates was determined using a luminometer. (B) Effect of NS5A expression on luciferase activity under the control of truncated IL-8 promoters. Ten micrograms of the relevant reporter plasmid was transfected into HeLa FL-NS5A-NR cells, and cells were grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.
FIG. 6
FIG. 6
Characterization of domains on NS5A and the IL-8 promoter required for transactivation. (A) Effect of NS5A mutant proteins on luciferase expression under the control of the full-length (546-luc) IL-8 promoter. Ten-microgram quantities of the 546-luc plasmid and pTRE-FL-NS5A-NR, NS5A-ΔN110, NS5A-ΔN222, or NS5A-ΔC117 were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. The data shown are the fold changes in luciferase activity with the gene in the induced versus the uninduced state. (B) Im- munofluoresence analysis of HeLa cells expressing FL-NS5A-NR, NS5A-ΔN222, or NS5A-ΔC117. HeLa Tet-Off cells stably transfected with FL-NS5A-NR (top panels), NS5A-ΔN222 (middle panels), or NS5A-ΔC117 (lower panels) were grown in the absence or presence of tetracycline (Tc) to induce or repress NS5A expression for 48 h. Cells were fixed and stained with a monoclonal NS5A antibody and then with Cy3-linked anti-mouse secondary antibodies, as described in Materials and Methods. Images are at a ×100 amplification. (C) Effect of NS5A-ΔN222 expression on luciferase activity under the control of mutated IL-8 promoters. Ten-microgram quantities of NS5A-ΔN222 and the relevant reporter plasmid were cotransfected into HeLa Tet-Off cells; cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated with 4 ng of TNF-α for 6 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. NF-κB–luc, AP-1–luc, and NF–IL-6–luc represent the 133-luc plasmid with point mutations in the NF-κB, AP-1, and NF–IL-6 binding sites.
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