Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines

Abstract

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.

FIG. 1.

Establishment of stable macrophage cell lines expressing HCV proteins. (A) Cell lysates were prepared from macrophage cell lines expressing each of the HCV proteins (4 × 10⁶ cells) and immunoblotted with antibodies against HCV proteins or β-actin. (B) Total RNA was extracted from macrophage cell lines expressing NS5A (gray bars) or control (white bars), and the expression of mRNA of TLRs was determined by real-time PCR. (C) The subcellular localization of NS5A was examined by confocal microscopy. Cells were fixed with 4% paraformaldehyde-PBS, permeabilized with 0.5% Triton X-100, and stained with specific antibodies. Cells expressing NS5A or control cells were extracted into cytosol (C), membrane-organelle (M), and nuclear (N) fractions. Each fraction was concentrated and subjected to immunoblotting with specific antibodies. PA28α, calregulin, and histone H1 were used as markers for cytosol, membrane-organelle, and nuclear fractions, respectively.

FIG. 2.

Expression of HCV nonstructural proteins modulates IL-6 production and MAPK cascades through the TLR-dependent signaling pathway in macrophage cell lines. (A) Cells were seeded onto 96-well plates (1 × 10⁵ cells/well) and stimulated with the indicated amounts of mCpG, R-837, LPS, or PGN. After 24 h of stimulation, IL-6 production in the culture supernatants was determined by sandwich ELISA. Data are shown as means ± standard deviations (SD). (B) Cells (2 × 10⁶ cells/well) were stimulated with 10 μg/ml of R-837 for the times indicated, and ERK1/2 phosphorylation was determined by immunoblotting with antibodies to ERK and phosphorylated ERK (p-ERK). Asterisks indicate nonspecific bands. (C) Cells (2 × 10⁶ cells/well) were stimulated with 10 μg/ml of mCpG, 25 ng/ml of LPS, or 10 μg/ml of PGN for the times indicated, and ERK1/2 phosphorylation was determined by immunoblotting.

FIG. 3.

Effect of NS5A expression on the production of cytokines and chemokines in response to TLR ligands in macrophage cell lines. Cells (3 × 10⁶ cells/well) were stimulated with 10 μg/ml of mCpG, 10 μg/ml of R-837, 25 ng/ml of LPS, and 10 μg/ml of PGN for the times indicated. Total RNA was extracted from macrophage cell lines expressing NS5A (gray bars) or control (white bars), and the expression of mRNA of IL-1α and Ccl2 (A) and IFN-β and IL-6 (B) was determined by real-time PCR.

FIG. 4.

TLR-dependent and -independent immune activation of macrophage cells expressing theNS3/4A or NS5A protein by RNA virus and dsRNA. (A) Myc-His-mTRIF was coexpressed with Flag-NS3, -NS3/4A, or -NS3/4A(S139A) in 293T cells and immunoblotted (IB) with antibodies against His and Flag. (B) Alignment of the flanking sequence of NS3 protease cleavage sites of NS4A/4B, NS4B/5A, TRIF, and IPS-1 of human and murine origins. The cleavage site is indicated by an arrow. (C) Flag-mIPS-1 and a mutant with Cys⁵⁰⁸ replaced with Ala (C508A) were coexpressed with Flag-NS3, -NS3/4A, or -NS3/4A(S139A) in 293T cells and immunoblotted with antibodies against mIPS-1 and NS3. (D) Processing of endogenous mIPS-1. Cell lysates of the macrophage cell lines expressing NS3, NS3/4A, and NS3/4A(S139A) were immunoblotted with antibodies against mIPS-1, NS3, and β-actin. The cleavage product of mIPS-1 is indicated as mIPS-I*. (E) Cells (3 × 10⁶ cells/well) were stimulated with 2 × 10⁵ PFU/ml of VSV or 50 μg/ml of poly(I:C) for the times indicated. Total RNA was extracted from the macrophage cell lines expressing NS3/4A (black bars), NS5A (gray bars), or control (white bars), and the expression of mRNA of IFN-β, IL-1α, IFN-α1, and IFN-α4 was determined by real-time PCR.

FIG. 5.

NS5A interacts with MyD88. MyD88-His was coexpressed with Flag-core/E1/E2 or -NS5A (A) or Flag-NS3, -NS3/4A, -NS4B, -NS5A, or -NS5B (B) in 293T cells; immunoprecipitated (IP) with anti-Flag, E1, or E2 antibody; and immunoblotted (IB) with anti-His antibody. (C) Flag-NS5A was coexpressed with MyD88-His, TRAM-His, TIRAP-His, or TRIF-HA in 293T cells and immunoprecipitated with anti-His or -HA antibody. The immunoprecipitates were immunoblotted with anti-Flag antibody. Asterisks indicate nonspecific bands.

FIG. 6.

NS5A interacts with the death domain of MyD88 through the ISDR and inhibits recruitment of IRAK to MyD88. (A) The structure of NS5A and the MyD88 binding region are indicated at the top. MyD88-His was coexpressed with C-terminal deletion mutants of Flag-NS5A in 293T cells, immunoprecipitated (IP) with anti-His antibody, and immunoblotted (IB) with anti-Flag antibody (left). MyD88-His was coexpressed with Flag-NS5A deletion mutants (Δ240-280 or Δ280-300) in 293T cells, immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-His antibody (right). (B) Flag-NS5A was coexpressed with N-terminal or C-terminal deletion mutants of MyD88-His (ΔN1, ΔN2, ΔN3, ΔC1, ΔC2, or ΔC3) in 293T cells, immunoprecipitated with anti-His antibody, and immunoblotted with anti-Flag antibody. The structures of MyD88 and the deletion mutants and the NS5A binding region are indicated on the left. (C) Flag-MyD88 (left) or Flag-NS5A (middle) was coexpressed with IRAK-1-Myc or IRAK-4-Myc in 293T cells, immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Flag antibody. Flag-MyD88 and IRAK-1-Myc were coexpressed with Flag-NS5A in 293T cells, immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Flag antibody. The effect of the increase in Flag-NS5A expression on the interaction of MyD88 with IRAK-1 was examined by transfection with 0.1, 0.5, or 2 μg of Flag-NS5A expression plasmid (right).

FIG. 7.

NS5A of other genotypes also interacts with MyD88 and inhibits the TLR signaling pathway. (A) Flag-NS5As of other genotypes were coexpressed with MyD88-His in 293T cells, immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (IB) with anti-His antibody. (B) The wild type or a deletion mutant lacking amino acids 240 to 280 of Flag-NS5A of genotype 1a or 2a was coexpressed with MyD88-His in 293T cells, immunoprecipitated with anti-Flag antibody, and immunoblotted with anti-His antibody. (C) Amino acid sequences of ISDR and its adjacent region of strains H77c (genotype 1a), J1 (genotype 1b), Con1 (genotype 1b), and JFH1 (genotype 2a). The conserved amino acids among genotypes 1a and 1b are indicated by boxes. Conserved amino acids among all strains are indicated by asterisks. (D) Macrophage cell lines expressing NS5A of genotypes 1a (H77c), 1b (J1), and 2a (JFH1) were established. Cells were stimulated with the indicated amounts of mCpG, R-837, LPS, or PGN, and the production of IL-6 in the culture supernatants was determined by ELISA 24 h after stimulation. Data are shown as the means ± SD.

FIG. 8.

ISDR in NS5A participates in the inhibition of the MyD88-dependent signaling pathway. (A) Structures of NS5A mutants lacking amino acid residues 240 to 280, in which the ISDR/MyD88-interacting region is located (Δ240-280), and lacking amino acid residues 280 to 300 (Δ280-300) (left). Immunoblot analyses of cells expressing wild-type or mutant NS5A (right) are shown. (B) Cells expressing wild-type or mutant NS5A were stimulated with the indicated amounts of mCpG, R-837, LPS, or PGN, and the production of IL-6 in the culture supernatants was determined by ELISA 24 h after stimulation. Data are shown as the means ± SD. (C) Phosphorylation of STAT1 or PKR in response to treatment with murine IFN-α or infection with VSV. The cell lines were stimulated with two doses of murine IFN-α (2 × 10³ and 2 × 10² units/ml) or VSV (2 × 10⁷ and 2 × 10⁶ PFU/ml). After 24 h of stimulation, cell extracts were immunoblotted (IB) with specific antibodies. Phosphorylated STAT1 and PKR and the total amounts of STAT1 and β-actin were determined. The asterisk indicates nonspecific bands.