Characterization of truncated forms of hepatitis C virus glycoproteins
Characterization of truncated forms of hepatitis C virus glycoproteins
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DOI：
10.1099/0022-1317-78-9-2299
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发表时间：
1997-09-01
影响因子：
3.8
通讯作者：
Dubuisson, J
中科院分区：
文献类型：
--
作者：
Michalak, JP;Wychowski, C;Dubuisson, J
丙型肝炎病毒 (HCV) 糖蛋白（E1 和 E2）均含有羧基末端疏水区，推测其充当膜锚。当它们在动物细胞培养物中表达时，这些糖蛋白以成熟复合物和错误折叠聚集体形式保留在内质网中。本研究检查了羧基末端缺失对 HCV 糖蛋白分泌和折叠的影响。构建了表达以氨基酸311、330、354和360(截短的E1)以及661、688、704和715(截短的E2)结尾的这些糖蛋白的截短形式的辛德毕斯和/或牛痘病毒重组体。当使用辛德毕斯病毒载体表达时，仅以氨基酸311结尾的E1和E2的截短形式(E1(t311)) 和 661 (E2(t661)) 分别被有效分泌。对痘苗病毒表达的 E2 糖蛋白的截短形式分泌的分析表明，对于像 E2(t715) 这样大的蛋白质，仍然观察到显着的分泌。然而，只有分泌型E2(t661)似乎被正确折叠，当E1(t311)和E2(t661)共表达时，在细胞培养物上清液中也检测到分泌型HCV糖蛋白复合物。然而，这些分泌的复合物以及单独表达的 E1(t311) 都被错误折叠。借助构象敏感单克隆抗体（对于 E2）或通过分析分子内二硫键形成（对于 E1）来评估 E1 和 E2 糖蛋白共表达对彼此折叠的影响。我们的数据表明，E2 的折叠独立于 E1，但 E2 是 E1 正确折叠所必需的。
Hepatitis C virus (HCV) glycoproteins (E1 and E2) both contain a carboxy-terminal hydrophobic region, which presumably serves as a membrane anchor, When they are expressed in animal cell cultures, these glycoproteins, in both mature complexes and misfolded aggregates, are retained in the endoplasmic reticulum, The effect of carboxyterminal deletions on HCV glycoprotein secretion and folding was examined in this study. Sindbis and/or vaccinia virus recombinants expressing truncated forms of these glycoproteins ending at amino acids 311, 330, 354 and 360 (truncated E1), and 661, 688, 704 and 715 (truncated E2) were constructed, When expressed using Sindbis virus vectors, only truncated forms of E1 and E2 ending at amino acids 311 (E1(t311)) and 661 (E2(t661)), respectively, were efficiently secreted. Analysis of secretion of truncated forms of E2 glycoprotein expressed by vaccinia viruses indicated that significant secretion was still observed for a protein as large as E2(t715). However, only secreted E2(t661) appeared to be properly folded, Secreted HCV glycoprotein complexes were also detected in the supernatant of cell culture when E1(t311) and E2(t661) were coexpressed. Nevertheless, these secreted complexes, as well as E1(t311) expressed alone, were misfolded. The effect of coexpression of E1 and E2 glycoproteins on each other's folding was evaluated with the help of a conformation-sensitive monoclonal antibody (for E2) or by analysing intramolecular disulfide bond formation (for E1). Our data indicate that the folding of E2 is independent of E1, but that E2 is required for the proper folding of E1.