Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease

J Biol Chem. 2005 May 27;280(21):20483-92. doi: 10.1074/jbc.M500422200. Epub 2005 Mar 14.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Vesicular Transport / chemistry*
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Amino Acid Sequence
  • Binding Sites
  • Circular Dichroism
  • Enzyme Inhibitors
  • Hepacivirus / enzymology
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Peptides / chemistry
  • Protein Structure, Secondary
  • Sequence Homology
  • Spectrometry, Fluorescence
  • Substrate Specificity
  • Viral Nonstructural Proteins / antagonists & inhibitors
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / metabolism*

Substances

  • Adaptor Proteins, Vesicular Transport
  • Enzyme Inhibitors
  • NS-5 protein, hepatitis C virus
  • NS3 protein, hepatitis C virus
  • Peptide Fragments
  • Peptides
  • TICAM1 protein, human
  • Viral Nonstructural Proteins
  • polyproline