HUMAN AND MURINE ANTIBODY RECOGNITION IS FOCUSED ON THE ATPASE HELICASE, BUT NOT THE PROTEASE DOMAIN OF THE HEPATITIS-C VIRUS NONSTRUCTURAL-3 PROTEIN/

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been show n to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two th irds is comprised of an adenosine triphosphatase (ATPase)/helicase dom ain. These domains are essential for the maturation of the carboxy-ter minal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses i nduced 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 w ith 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 do main of NS3. Human antibodies reactive with NS3-FL were highly restric ted to the immunoglobulin G1 (IgG1) isotype and were inhibited by solu ble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATP ase/helicase) reactivity decreased on denaturation by sodium dodecyl s ulfate (SDS) and p-mercaptoethanol (2ME), suggesting the recognition o f nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary ant ibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS 3 protease response was not observed after primary or secondary immuni zations. Thus, the human and murine humoral immune responses to the HC V NS3 protein are focused on the ATPase/helicase domain, are restricte d to the IgG1 isotype in humans, and are conformationally dependent. U nexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immun ogenicity in terms of antibody production.