Chen M, Sällberg M, Sönnerborg A, Jin L, Birkett A, Peterson D, Weiland O, Milich D R
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Hepatology. 1998 Jul;28(1):219-24. doi: 10.1002/hep.510280128.
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.
丙型肝炎病毒（HCV）非结构（NS）3蛋白已被证明至少具有两个酶结构域。氨基末端三分之一包含一个丝氨酸蛋白酶结构域，而羧基末端三分之二由一个三磷酸腺苷酶（ATPase）/解旋酶结构域组成。这些结构域对于HCV多蛋白羧基末端部分的成熟至关重要，并催化RNA基因组的帽合成。在本报告中，使用从大肠杆菌中表达和纯化的重组全长NS3（NS3-FL）蛋白，或分离的蛋白酶或ATPase/解旋酶结构域，对NS3诱导的人和小鼠抗体反应进行了表征。在酶联免疫吸附测定（EIA）中检测了40例慢性HCV感染患者的血清与NS3蛋白组的抗体结合情况。几乎所有患者血清都含有针对NS3-FL和ATPase/解旋酶结构域的特异性抗体，而只有10%的血清与NS3的蛋白酶结构域发生反应。与NS3-FL反应的人抗体高度局限于免疫球蛋白G1（IgG1）同种型，并被可溶性ATPase/解旋酶抑制，但不被蛋白酶结构域抑制。抗NS3（ATPase/解旋酶）反应性在十二烷基硫酸钠（SDS）和β-巯基乙醇（2ME）变性后降低，表明识别非线性或构象性B细胞决定簇。与受感染的人类相似，用NS3-FL免疫的小鼠对NS3 ATPase/解旋酶结构域产生了高滴度的初次抗体反应，而在初次或二次免疫后未观察到抗NS3蛋白酶反应。因此，人和小鼠对HCV NS3蛋白的体液免疫反应集中在ATPase/解旋酶结构域，在人类中局限于IgG1同种型，并且依赖于构象。出乎意料的是，在这两个物种中，全长NS3背景下的NS3蛋白酶结构域在抗体产生方面似乎具有较低的固有免疫原性。
