title: Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease.
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.
authors.affiliation: Department of Microbiology and Immunology, Center for Hepatitis Research, Institute for Human Infections and Immunity, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
authors.forename: Josephine C
authors.initials: JC
authors.lastname: Ferreon
authors.forename: Allan Chris M
authors.initials: AC
authors.lastname: Ferreon
authors.forename: Kui
authors.initials: K
authors.lastname: Li
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004791:Enzyme Inhibitors; D016174:Hepacivirus / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017385:Sequence Homology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010455:Peptides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C470409:TICAM1 protein, human; C011083:polyproline; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase