Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Feb 22;102(8):2992-7.
doi: 10.1073/pnas.0408824102. Epub 2005 Feb 14.

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

Kui Li  1 , Eileen Foy, Josephine C Ferreon, Mitsuyasu Nakamura, Allan C M Ferreon, Masanori Ikeda, Stuart C Ray, Michael Gale Jr, Stanley M Lemon
Affiliations

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

Kui Li et al. Proc Natl Acad Sci U S A. .

Abstract

Toll-like receptors (TLRs) bind pathogen-specific ligands early in infection, initiating signaling pathways that lead to expression of multiple protective cellular genes. Many viruses have evolved strategies that block the effector mechanisms induced through these signaling pathways, but viral interference with critical proximal receptor interactions has not been described. We show here that the NS3/4A serine protease of hepatitis C virus (HCV), a virus notorious for its ability to establish persistent intrahepatic infection, causes specific proteolysis of Toll-IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF or TICAM-1), an adaptor protein linking TLR3 to kinases responsible for activating IFN regulatory factor 3 (IRF-3) and NF-kappaB, transcription factors controlling a multiplicity of antiviral defenses. NS3/4A-mediated cleavage of TRIF reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF-kappaB. This uniquely broad mechanism of immune evasion potentially limits expression of multiple host defense genes, thereby promoting persistent infections with this medically important virus.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
TRIF is a substrate for NS3/4A protease. (A) Majority-rule consensus sequences for NS3/4A trans-cleavage sites within the genotype 1b HCV polyprotein, compared with candidate cleavage sites in TLR3, TRIF, IKKε, and TBK1. Conserved Cys–(Ser/Ala) and P6 acidic residues are highlighted. Residues aligning with those in the consensus viral substrates are shown in boldface; viral residues aligning with the potential cleavage site at Cys-372 of TRIF are underlined. (B) TLR3, TRIF, IKKε, and TBK1 synthesized in reticulocyte lysate were incubated with MB–scNS3 or protease-deficient C1125A MB–scNS3 before SDS/PAGE. TRIF cleavage was inhibited by SCH6 (10 μM), a specific ketoamide inhibitor of the protease. (C) Coomassie blue-stained SDS/PAGE gel of reactions containing recombinant TRIF and either scNS3 or NS3p/4A. N-terminal amino acid sequencing confirmed the identity of the ΔN372 product. (D) Cleavage of a TRIF peptide (PPPPPPPPSSTPCSAHL) by scNS3 protease. Cleavage products were analyzed by HPLC at 0, 20, 115, and 235 min. (E) Domain map of TRIF showing the location of NS3/4A cleavage relative to sites of interaction with the TIR domain and recognized signaling partners.
Fig. 2.
Fig. 2.
NS3/4A mediates in vivo cleavage of TRIF. (A) HEK 293 cells were transfected with vectors expressing N-terminally tagged myc-TRIF or the cleavage-resistant mutant myc-C372R TRIF, NS3/4A (or empty vector), and GFP as a transfection control. (B) Pulse–chase labeling of myc-TRIF expressed in HEK 293 cells in the presence or absence of NS3/4A coexpression. IVT, in vitro synthesized TRIF; Mock, mock-transfected cells. (C) Expression of TRIF-Flag and GFP from cotransfected plasmids in HCV RNA-bearing Huh7 2-3 cells and their IFN-cured progeny, 2-3c cells. TRIF-Flag abundance was quantified relative to GFP by densitometry. (D) Immunoblots showing endogenous TRIF abundance (lanes 1 and 2) in HeLa SL1 cells containing a replicating subgenomic RNA and IFN-cured SL1c cells. SL1c cells in lane 3 were transfected with a vector expressing TRIF to mark its position in SDS/PAGE.
Fig. 3.
Fig. 3.
TRIF proteolysis inhibits pIC-induced activation of IFN-β and PRDII promoters and ISG expression in UNS3-4A-24 cells that conditionally express NS3/4A. (A) Immunoblots showing abundance of transfected TRIF-Flag (Upper) and the cleavage-resistant myc-TRIF-C372R mutant (Lower) with or without NS3/4A expression. (B) IFN-β (Left) and PRDII (Right) promoter activities 8 h after addition of pIC to the media (50 μg/ml). For comparison, IFN-β promoter activity is also shown 16 h after Sendai virus (SenV) infection (Center). Where indicated, cells were treated with 10 μM SCH6. (C) Immunoblot detection of IRF-3, ISG15, and ISG56 after pIC exposure (10 h) or SenV infection (16 h). (D) ISG15 and ISG56 expression with (lanes 5–8) and without (lanes 1–4) NS3/4A expression at various times after the addition of pIC to media. (E) (Left) pIC stimulation of the IFN-β promoter is inhibited by RNA interference silencing of TLR3 and TRIF and, to a lesser extent, RIG-I. Numbers over each column pair indicate the fold-induction of promoter activity by pIC. Ctrl, control siRNA. (Upper Right) RT-PCR assays showing that RNA interference silencing of TLR3 and TRIF, but not RIG-I, prevents pIC induction of ISG56 and RANTES transcription. (Lower Right) RT-PCR detection of TLR3, TRIF, and RIG-I mRNAs after transfection of specific siRNAs. Cells were cultured in the presence of tetracycline (no NS3/4A expression). (F) EMSA demonstrating that NS3/4A expression attenuates pIC stimulated NF-κB binding to the PRDII element. IL-1, IL-1 treatment control; M, mock-treated.
Fig. 4.
Fig. 4.
The TLR3 pathway is compromised in HeLa SL1 cells containing replicating HCV RNA but not in cured SL1c cells. (A) IFN-β (Upper) and RANTES (Lower) promoter activities in SL1 and SL1c cells, after exposure to extracellular pIC (50 μg/ml) (Left) or SenV infection (Right). (B) Ectopic expression of NS3/4A blocks activation of the IFN-β promoter in SL1c cells exposed to pIC. V, empty vector; 3/4A, NS3/4A; S139A, an active site mutant of NS3/4A. Where indicated, cells were treated with 10 μM SCH6. (C) IFN-β promoter activity in SL1c cells transfected with siRNAs targeting TLR3, TRIF, or RIG-I, with and without pIC stimulation. Ctrl, control siRNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Seeff, L. B. (1997) Hepatology 26, 21S-28S. - PubMed
    1. Racanelli, V. & Rehermann, B. (2003) Trends Immunol. 24, 456-464. - PubMed
    1. Foy, E., Li, K., Wang, C., Sumter, R., Ikeda, M., Lemon, S. M. & Gale, M., Jr. (2003) Science 300, 1145-1148. - PubMed
    1. Hiscott, J., Pitha, P., Genin, P., Nguyen, H., Heylbroeck, C., Mamane, Y., Algarte, M. & Lin, R. (1999) J. Interferon Cytokine Res. 19, 1-13. - PubMed
    1. Yoneyama, M., Suhara, W. & Fujita, T. (2002) J. Interferon Cytokine Res. 22, 73-76. - PubMed

Publication types

MeSH terms

LinkOut - more resources