Matsuura Y, Suzuki T, Suzuki R, Sato M, Aizaki H, Saito I, Miyamura T
Department of Virology II, National Institute of Health, Tokyo, Japan.
Virology. 1994 Nov 15;205(1):141-50. doi: 10.1006/viro.1994.1629.
Processing of the envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV) was investigated by using cDNA clones covering the structural and part of the nonstructural (NS) protein regions. The cDNA clones expressed in mammalian and insect cells were immunoprecipitated by serum of a hepatitis C patient and by monoclonal and polyclonal antibodies raised against the recombinant proteins expressed in insect cells or Escherichia coli. The E2 protein expressed in both insect and mammalian cells was a glycoprotein of 60 kDa (gp60) and removal of the sugar residues by N-glycanase yielded 38- and 40-kDa proteins. Pulse-chase experiments revealed that efficient expression and processing of the envelope proteins required coexpression with the flanking core and NS2 proteins. Not only E1 and E2 proteins but also NS2 and NS3 proteins were coprecipitated by anti-E1 or anti-E2 monoclonal antibody in the cells infected with the recombinant baculovirus expressing structural and NS proteins (NS2 and NS3), while only the NS3 protein was precipitated by anti-NS3 antibody. The association of E1 and E2 proteins was not influenced by the presence of a reducing agent and was still observed in the cells coinfected with the deletion mutants lacking both internal and C-terminal hydrophobic regions of each protein. Furthermore, the truncated forms of the E1 and E2 proteins were secreted into the culture supernatant and some of them were still associated with each other.
利用覆盖丙型肝炎病毒（HCV）结构蛋白和部分非结构（NS）蛋白区域的cDNA克隆，对HCV包膜糖蛋白（E1和E2）的加工过程进行了研究。在哺乳动物细胞和昆虫细胞中表达的cDNA克隆，用丙型肝炎患者的血清以及针对在昆虫细胞或大肠杆菌中表达的重组蛋白产生的单克隆和多克隆抗体进行免疫沉淀。在昆虫细胞和哺乳动物细胞中表达的E2蛋白是一种60 kDa的糖蛋白（gp60），用N-糖苷酶去除糖基后产生38 kDa和40 kDa的蛋白。脉冲追踪实验表明，包膜蛋白的有效表达和加工需要与侧翼的核心蛋白和NS2蛋白共表达。在用表达结构蛋白和NS蛋白（NS2和NS3）的重组杆状病毒感染的细胞中，抗E1或抗E2单克隆抗体不仅能共沉淀E1和E2蛋白，还能共沉淀NS2和NS3蛋白，而抗NS3抗体只能沉淀NS3蛋白。E1和E2蛋白的结合不受还原剂存在的影响，在共感染缺失每种蛋白内部和C末端疏水区域的缺失突变体的细胞中仍能观察到。此外，E1和E2蛋白的截短形式分泌到培养上清中，其中一些仍相互结合。
