Biological function of hepatitis C virus nonstructural protein NS3

丙型肝炎病毒是慢性丙型肝炎的病原体。利用重组DNA技术对其进行了克隆，推测其为黄病毒科的一员。然而，病毒非结构蛋白NS3在病毒复制中的活性和意义尚不清楚。1.构建了含NS3 5‘区(pHCN3-N’)和3‘区(pHCN3-C’)的丙型肝炎病毒NS3表达载体，并在1 mM IPTG存在下表达了丙型肝炎病毒NS3相关蛋白。此外，提取的蛋白仅与丙型肝炎患者血清呈阳性反应，与健康对照血清无反应。对丙型肝炎患者进行了HCN3-C‘反应性检测，经干扰素治疗后转为阴性。此外，在丙型肝炎病毒诱导的肝病中，反应性随病程进展而增加。用该蛋白免疫兔，获得抗HCN3-C‘的抗血清。取1例慢性丙型肝炎患者的肝组织进行匀浆。抗血清与肝组织之间有明显的免疫反应，蛋白大小为70 kDa。采用SDS-PAGE和电洗脱法对HCN3-C‘蛋白进行纯化。在纯化的蛋白中，可以检测到显著的ATPas活性。本研究表明，至少部分地，丙型肝炎病毒NS3在丙型肝炎的进展中起作用。此外，还提出了丙型肝炎病毒NS3在病毒复制中的意义。

Hepatitis C virus (HCV) is the causative agent of chronic type C hepatitis. It is cloned by the recombinant DNA techniques and suggested to be a member of the family Flaviviridae. However, the activity and significance of virus nonstructural protein NS3 on the replication of virus are not known. To clarify these, HCV-NS3 related proteins were extracted and their function was tested as follows :1. HCV-NS3 expression vectors were prepared containing either 5'(pHCN3-N') region of NS3 or 3'(pHCN3-C') and HCV-NS3 related proteins were expressed in the bacteria in the presence of 1mM IPTG.When the expressed proteins were reacted with human sera, HCN3-N' protein which was produced in the bacteria containing pHCN3-N' was found to be the size of 32 kDa and HCN3-C' from pHCN3-C' was of 49 kDa, respectively. In addition, the reaction was positive only with sera of patients with hepatitis C.No reaction was seen between the extracted proteins and sera of healthy controls.2. The reactivity of HCN3-C' was tested in patients with hepatitis C and found that it became to be negative after the treatment of the patients with interferon. In addition, the reactivity was increased as progressed in the HCV-induced liver disease.3. Antisera against HCN3-C' were obtained following the immunization of rabbits with the protein. Liver tissue, which was taken by needle biopsy of a patient with chronic type C hepatitis, was homogenized. Between the antisera and the liver tissue, a clear immunoreaction was found in a size of 70 kDa protein.4. The protein HCN3-C' was purified by SDS-PAGE and the electro-elution method. In the purified protein, it was possible to detect a remarkable activity of ATPase.The present study suggests that HCV-NS3 plays, at least in part, a role in the progession of type C hepatitis. In addition, the significance of HCV-NS3 in the replication of virus is suggested.