Molecular Determinants of TRIF Proteolysis Mediated by the Hepatitis C Virus NS3/4A Protease

Stanley M Lemon,Josephine C Ferreon,Kui Li,Allan Chris M Ferreon

doi:10.1074/jbc.m500422200

Abstract

Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses. HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its proline-rich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-beta (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P(6) position in lieu of the acidic residue present in viral substrates. Circular dichroism (CD) spectroscopy confirmed that a substantial fraction of TRIF exists as polyproline II helices, and inclusion of the polyproline track increased affinity of P side TRIF peptides for the HCV-BK protease. A polyproline II peptide representing an SH3 binding motif (PPPVPPRRR, Sos) bound NS3 with moderate affinity, resulting in inhibition of proteolytic activity. Chemical shift perturbations in NMR spectra indicated that Sos binds a 3(10) helix close to the protease active site. Thus, a polyproline II interaction with the 3(10) helix likely facilitates NS3/4A recognition of TRIF, indicating a significant difference from NS3/4A recognition of viral substrates. Because SH3 binding motifs are also present in NS5A, a viral protein that interacts with NS3, we speculate that the NS3 3(10) helix may be a site of interaction with other viral proteins.

Highlights

Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses
HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its prolinerich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-␤ (TRIF)
TRIF Is a Novel Host Substrate for the HCV NS3/4A Protease—NS3 is a bifunctional protein, with a serine protease domain located within its N-terminal third, and ATPase/RNA helicase activity located within its C-terminal two-thirds [34]

Summary

EXPERIMENTAL PROCEDURES

Peptides—Synthetic peptides representing sequences spanning the NS3/4A cleavage sites at the viral NS5A/5B (H-EDVV␣C/SMSY-OH) [26], NS4A/4B (H-EFDEMEEC/ASHLPYI-OH), and NS4B/5A (H-ECTTPC/SGSWLRD-OH) junctions, the NS3/4A cleavage site within TRIF (Ac-PSSTPC/SAHL-amide, Ac-PPPPPPPPSSTPC/SAHL-amide) [13], their P side cleavage products (Ac-PSSTPC-OH, and Ac-PPPPPPPPSSTPC-OH), and an HCV peptide suitable for use in a fluorescence resonance energy transfer (FRET) assay (Ac-DED(EDANS)EEAbu␺ [COO]ASK-(DABCYL)-am) [27] were purchased from Anaspec and were Ͼ95% pure. Estimated EC50 for scNS3 Cleavage of Full-length TRIF—To determine the enzyme concentration required to hydrolyze 50% of the fulllength TRIF protein (EC50), different reaction tubes were prepared with 4 ␮M TRIF and varying scNS3 concentrations (0.25, 0.5, 1, 2, 3, 5 ␮M) in 20 mM Tris, 30 mM DTT, pH 7.5. The data (TRIF intensity versus enzyme concentration) was fitted in a nonlinear fashion to the following simplified four-parameter logistic function [27] derived from the Hill equation, y ϭ 100/͑1 ϩ͑E/EC50)n (Eq 2). Sensitivity-enhanced 1H-15N HSQC spectra were recorded with identical acquisition parameters and 0.5 mM 15N-labeled scNS3 protein (75 mM potassium phosphate, 5% glycerol, 25 mM DTT, 0.015% NaN3, 10% D2O, pH 6.5) with different Sos peptide concentrations (0, 0.3, 1.3, 2.2 mM final). Visualizations—The Molmol program [32] was used to generate ribbon models of the NS3 protease (PDB entry 1NS3) (see Fig. 7) and the ball-and-stick model of the Sos peptide (see Fig. 4B), adopted from the co-crystal structure of the SEM-5 C-terminal SH3 domain with the Sos peptide (PDB entry 1SEM, chain C) [33]

RESULTS

E F DEMEE C

DISCUSSION