Opstelten D J, de Groote P, Horzinek M C, Rottier P J
Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, The Netherlands.
Arch Virol Suppl. 1994;9:319-28. doi: 10.1007/978-3-7091-9326-6_32.
Coronaviruses are assembled by budding into pre-Golgi membranes. Using different approaches we have demonstrated that the spike (S) protein and the membrane (M) protein of mouse hepatitis virus (MHV) associate to form large complexes. Newly synthesized M was found in these complexes almost immediately after its synthesis, whereas the S protein started to appear in heterocomplexes after 10-20 min. This is consistent with the slow rate of folding of S and with the observation that folding of S preceeds its association with M. While the folding of S involves the formation of multiple disulfide bonds, folding of M is disulfide-independent. This contrast was reflected by the differential sensitivity of the two proteins to reduction with dithiothreitol (DTT). Addition of DTT to the culture medium of MHV-infected cells drastically impaired the folding of S, but not of M. Consequently, the S protein was unable to interact with M. Under these conditions, S stayed in the ER while M was transported efficiently beyond the site of budding to the Golgi complex. We conclude that the association of S with M is an essential step in the formation of the viral envelope and in the accumulation of both proteins at the site of virus assembly.
冠状病毒通过出芽进入高尔基体前膜进行组装。我们采用不同方法证明，小鼠肝炎病毒（MHV）的刺突（S）蛋白和膜（M）蛋白相互作用形成大的复合物。新合成的M在合成后几乎立即出现在这些复合物中，而S蛋白在10 - 20分钟后开始出现在异源复合物中。这与S蛋白缓慢的折叠速率一致，也与S蛋白在与M蛋白结合之前先进行折叠的观察结果相符。虽然S蛋白的折叠涉及多个二硫键的形成，但M蛋白的折叠不依赖二硫键。这一差异反映在两种蛋白对二硫苏糖醇（DTT）还原作用的不同敏感性上。向感染MHV的细胞培养基中添加DTT会严重损害S蛋白的折叠，但不会影响M蛋白的折叠。因此，S蛋白无法与M蛋白相互作用。在这种情况下，S蛋白滞留在内质网中，而M蛋白则有效地运输到出芽位点之外，到达高尔基体复合体。我们得出结论，S蛋白与M蛋白的结合是病毒包膜形成以及两种蛋白在病毒组装位点积累的关键步骤。
