title: Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein.
abstract: The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and beta-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Sällberg
authors.forename: A
authors.initials: A
authors.lastname: Sönnerborg
authors.forename: L
authors.initials: L
authors.lastname: Jin
authors.forename: A
authors.initials: A
authors.lastname: Birkett
authors.forename: D
authors.initials: D
authors.lastname: Peterson
authors.forename: O
authors.initials: O
authors.lastname: Weiland
authors.forename: D R
authors.initials: DR
authors.lastname: Milich
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity / Q000502:physiology; D004265:DNA Helicases / Q000235:genetics*; D010450:Endopeptidases / Q000235:genetics*; D016174:Hepacivirus / Q000378:metabolism*; D018937:Hepatitis C Antibodies / Q000276:immunology*; D019698:Hepatitis C, Chronic / Q000276:immunology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008968:Molecular Conformation; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
chemical_list: D018937:Hepatitis C Antibodies; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases