Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKKepsilon - PubMed (original) (raw)

Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKKepsilon

Adrien Breiman et al. J Virol. 2005 Apr.

Abstract

Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-beta via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKKepsilon and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF(-/-) mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKKepsilon, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-beta promoter transactivation, with IKKepsilon displaying the highest efficiency. Subsequently, overexpression of IKKepsilon resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-beta indicates that IKKepsilon expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.

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Figures

FIG. 1.

FIG. 1.

IFN production is inhibited in an HCV replicon-expressing cell. (A) Huh-7 Rep cells were plated at 20,000 cells/well in Labtek chambers. After 24 h of incubation, the cells were fixed with 3.7% paraformaldehyde, permeabilized with Triton X-100, and analyzed by immunofluorescence for the presence of HCV proteins with a monoclonal anti-NS3 (a gift from D.Moradpour) and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibodies or monoclonal anti-NS5A (Biodesign) and Texas Red-conjugated anti-mouse immunoglobulin antibodies. (B) Huh-7 and Huh-7 Rep cells were plated in six-well plates at 800,000 cells/well and 106 cells/well, respectively. At 24 h, the cells were infected with Sendaï virus (SeV) at 40 hemagglutination units (HAU)/well. At 4, 8, and 24 h after infection, infected and control (Cont) cells were washed twice with phosphate-buffered saline, RNA was extracted with RNABle, and the samples were processed for real-time RT-PCR analysis as described in Materials and Methods. For each sample, reverse transcription was conducted in the simultaneous presence of the 3′ primers for GAPDH and IFN-β to minimize errors. All values were normalized with GAPDH. (C) Huh-7 and Huh-7 Rep cells were transfected with 1 μg of IFN-β-pGL3 and 0.4 μg of a plasmid expressing Rous sarcoma virus β-galactosidase for normalization. After 8 h, cells were further infected with Sendaï virus at 40 hemagglutination units (HAU)/ml. After 24 h, the cell extracts were analyzed for luciferase activity in duplicate, andthe results are expressed as stimulation (n = fold) of luciferase activity after β-galactosidase normalization.

FIG. 2.

FIG. 2.

HCV NS3/4A protease does not affect IKKɛ/TBK-1-mediated IRF-3 phosphorylation. HEK 293T cells were seeded at 3.5 × 106 cells/100-mm plate and transfected after 24 h with Lipofectamine 2000 with 5 μg of plasmid expressing IRF-3 in the presence of (A) different concentrations of a plasmid expressing TBK-1, in either the absence or the presence of 15 μg of plasmids expressing NS3/4A or NS5A; (B) different concentrations of a plasmid expressing wild-type IKKɛ, either in the absence or in the presence of 15 μg of plasmids expressing NS3/4Aor NS3 alone; (C) different concentrations of a plasmid expressing wild-type IKK, in the presence of 15 μg of plasmid expressing NS5A; or (D) different amounts of plasmids expressing NS3/4A, NS5A, wild-type IKKɛ, or IKKɛ K38A. Cell extracts were prepared 24 h after transfection, and equivalent amounts of protein extract (45 μg) were used for immunoblot analysis.

FIG. 3.

FIG. 3.

NS3/4A affects IFN signaling via TRIF-dependent and TRIF-independent pathways. (A) HEK 293T cells were cotransfected with the IFN-β-pGL3 reporter construct and the PRLTK internal control in the presence or absence of NS3/4A and TRIF expression plasmids as described in Materials and Methods. (B) HEK 293 T cells were cotransfected with an empty vector (pcDNA3/Amp) or plasmid expressing TRIF (1.2 μg) alone or in the presence of 2,5 μg of plasmid expressing either NS3/4A or NS5A; 24 h after the transfection, RNA was extracted with RNABle, and the samples were processed for real-time RT-PCR analysis as described in Materials and Methods and for Fig. 1B. (C) Wild-type MEFs (white bars) and TRIF−/− MEFs (dark bars) were transfected with the IFN-β-pGL3 reporter construct and the PRLTK internal control in the presence or absence of NS3/4A and NS5A expression plasmids. Cells were either infected with Sendai virus or left untreated. Analysis of luciferase activity was measured 24 h after transfection and normalized with the Renilla luciferase activities. An immunoblot shows the presence of TRIF in the wild-type MEFs (+/+) and its absence in the TRIF−/− MEFs. (D) HEK 293 T cells were cotransfected with the IFN-β-pGL3 reporter construct (100 ng) and the PRLTK internal control in the presence or absence of 300 ng of ΔRIG-1, 300 ng of a dominant negative form of IKKɛ deprived of its catalytic domain (IKKɛ ΔC), and 300 ng of NS3/4A or NS5A expression plasmid. When indicated, the cells were infected with 40 hemagglutination units (HAU) of Sendai virus/106 cells for 16 h.

FIG. 4.

FIG. 4.

Relative efficiency of different components of IFN signaling to restore activation of the IFN-β promoter in the HCV replicon cells. (A and B) Huh-7 (A) and Huh-7 Rep (B) cells were transfected with 1 μg of IFN-β-pGL3, 1 μg of Rous sarcoma virus β-galactosidase as such or in the presence of 0.5 μg of IKKɛ, TBK-1, TRIF, RIG-I, or ΔRIG-I. The stimulation of luciferase activity was analyzed without (empty bars) or after infection with Sendaï virus at 40 hemagglutination units (HAU)/ml (grey bars). Analysis of luciferase activity was performed as described above. (C) Huh-7 cells and Huh-7 Rep cells were transfected with 5 μg of plasmids expressing IRF-3, wild-type IKKɛ, or IKKɛ K38A, as indicated. At 24 h after transfection, the cells were infected with 80 hemagglutination units (HAU)/ml of Sendai virus for 8 h. Cell extracts were prepared and equivalent amounts of protein extract (30 μg) were used for immunoblot analysis.

FIG. 5.

FIG. 5.

Overexpression of wild-type IKKɛ inhibits replication of an HCV replicon and partially restores IFN induction. Huh-7 Rep cells were plated in 100-mm plates at 2 × 106 cells/dish and transfected 24 h after seeding with 10 μg of an empty vector (Cont) or vectors expressing wild-type IKKɛ or IKKɛ K38A. At 24 h (1), 48 h (2), and 72 h (3) after transfection, RNA was extracted with RNABle, and the samples were processed for real-time RT-PCR analysis of HCV positive- (A) or negative-strand (B) RNA and of IFN-β RNA (C) as described in Materials and Methods. For each sample, reverse transcription was conducted in the simultaneous presence of the 3′ primers for GAPDH and HCV positive strand (A), GAPDH and HCV negative strand (B), and GAPDH and IFN-β (C) to minimize errors. The effect of IKKɛ on HCV RNA expression was analyzed in two independent experiments, and the results are expressed as the mean percent HCV RNA expression.

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