Op De Beeck Anne, Voisset Cécile, Bartosch Birke, Ciczora Yann, Cocquerel Laurence, Keck Zhenyong, Foung Steven, Cosset François-Loïc, Dubuisson Jean
CNRS-UPR2511, Institut de Biologie de Lille and Institut Pasteur de Lille, Lille, France.
J Virol. 2004 Mar;78(6):2994-3002. doi: 10.1128/jvi.78.6.2994-3002.2004.
Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.
丙型肝炎病毒（HCV）编码两种包膜糖蛋白，E1和E2，它们组装成非共价异二聚体，主要保留在内质网中。由于组装成颗粒以及从细胞中分泌会导致病毒包膜蛋白发生结构变化，因此有必要对与病毒粒子相关的蛋白进行表征，以便更好地了解它们如何成熟并在病毒进入过程中发挥功能。目前尚无高效可靠的细胞培养系统来扩增HCV，因此与病毒粒子相关的包膜糖蛋白尚未得到表征。最近，通过在逆转录病毒核心颗粒上展示未修饰的HCV包膜糖蛋白成功生成了感染性假型颗粒。由于HCV假型颗粒含有功能完全的包膜糖蛋白，这些包膜蛋白，或至少其中一部分，应处于与天然HCV颗粒上相似的成熟构象。在本研究中，我们使用构象依赖性单克隆抗体来表征与HCV假型颗粒相关的包膜糖蛋白。我们发现功能单元是一个包含复杂或杂合型聚糖的非共价E1E2异二聚体。在这些包膜糖蛋白通过分泌途径运输过程中，我们没有观察到任何细胞内蛋白酶介导成熟的证据。这些包膜糖蛋白被一组构象依赖性单克隆抗体以及参与HCV进入的分子CD81所识别。与HCV假型颗粒相关的功能性包膜糖蛋白也显示对低pH处理敏感。这种构象变化可能是启动融合所必需的。
