title: Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.
abstract: BACKGROUND
  It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation.
  
  
  METHODS
  Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined.
  
  
  RESULTS
  HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4.
  
  
  CONCLUSIONS
  C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Mawatari
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Uto
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Ido
authors.affiliation: Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Nakashima
authors.affiliation: Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Kanmura
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Kumagai
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Tabu
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tamai
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Moriuchi
authors.affiliation: Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Kagoshima, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Oketani
authors.affiliation: Miyazaki Prefectural Industrial Support Foundation, Miyazaki, Miyazaki, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Shimada
authors.affiliation: Kamakura Research Division, Chugai Pharmaceutical, Co. Ltd., Kamakura, Kanagawa, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sudoh
authors.affiliation: Division of Microbiology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Shoji
authors.affiliation: Department of HGF Tissue Repair and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Tsubouchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D003167:Complement Activation / Q000187:drug effects; D003181:Complement C4 / Q000737:chemistry / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012756:Sheep; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D003181:Complement C4; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C486464:telaprevir