FORMATION AND INTRACELLULAR-LOCALIZATION OF HEPATITIS-C VIRUS ENVELOPE GLYCOPROTEIN COMPLEXES EXPRESSED BY RECOMBINANT VACCINIA AND SINDBIS VIRUSES
FORMATION AND INTRACELLULAR-LOCALIZATION OF HEPATITIS-C VIRUS ENVELOPE GLYCOPROTEIN COMPLEXES EXPRESSED BY RECOMBINANT VACCINIA AND SINDBIS VIRUSES
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DOI：
10.1128/jvi.68.10.6147-6160.1994
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发表时间：
1994-10-01
影响因子：
5.4
通讯作者：
RICE, CM
中科院分区：
文献类型：
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作者：
DUBUISSON, J;HSU, HH;RICE, CM
丙型肝炎病毒（HCV）编码两种假定的病毒粒子糖蛋白（E1和E2），它们通过信号肽酶裂解从多蛋白中释放出来。在本报告中，我们对两种不同HCV菌株（H和BK）的E1和E2之间形成的复合物（称为E1E2）进行了表征，并研究了它们在细胞内的定位。利用牛痘病毒和Sindbis病毒载体在HepG2、BHK-21和PK-15三种不同细胞系中表达HCV结构蛋白。通过脉冲追踪分析和E2与抗E1单克隆抗体的共沉淀研究，E1与E2的结合动力学表明，稳定的E1E2复合物的形成是缓慢的。H株E1和E2达到半最大关联所需时间为60 ~ 85 min， BK株为165 min以上。在非离子洗涤剂的存在下，检测到两种形式的E1E2配合物。主要形式是由非共价相互作用稳定的E1和E2的异二聚体。一小部分由非均相的二硫化物连接的聚集体组成，这很可能代表错误折叠的复合物。通过获取内糖苷酶H抗性、亚细胞分离、免疫荧光、细胞表面免疫染色和免疫电镜检查HCV糖蛋白的翻译后加工和定位。没有观察到含有复杂n链聚糖的HCV糖蛋白，也没有在细胞表面检测到蛋白质。相反，这些蛋白质主要定位于内质网状网络，这表明它们保留在内质网状网络中存在某种机制。
Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.