Raamsman M J, Locker J K, de Hooge A, de Vries A A, Griffiths G, Vennema H, Rottier P J
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Institute of Virology, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands.
J Virol. 2000 Mar;74(5):2333-42. doi: 10.1128/jvi.74.5.2333-2342.2000.
The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.
最近研究表明，小囊膜（E）蛋白在冠状病毒的组装过程中发挥着至关重要的作用。表达研究显示，实际上病毒囊膜的形成仅需要E蛋白和膜（M）蛋白。由于人们对这种普遍低丰度的病毒粒子组分了解甚少，我们对小鼠肝炎病毒A59株（MHV - A59）的E蛋白进行了特性分析，该蛋白是一种由83个氨基酸残基组成的多肽。利用针对这种原本疏水的蛋白亲水性羧基末端的抗血清，我们发现E蛋白在受感染细胞中的合成动力学与其他病毒结构蛋白相似。无论是在感染过程中还是使用痘苗病毒表达系统单独表达时，该蛋白似乎都相当稳定。与缺乏预测的切割位点一致，发现该蛋白无需切割信号肽的参与即可整合到膜中，也未观察到该多肽的任何其他修饰。对表达E蛋白的细胞进行免疫荧光分析表明，亲水性尾部暴露在细胞质一侧。因此，该蛋白的这一结构域在病毒粒子外部无法检测到，但似乎在内部，受到蛋白水解降解的保护。这些结果得出了一个拓扑模型，即该多肽埋于膜内，跨脂质双层一次，可能两次，仅暴露其羧基末端结构域。最后，电子显微镜研究表明，细胞中E蛋白的表达诱导形成了在MHV - A59感染细胞中也观察到过的特征性膜结构，这些结构显然由大量管状、光滑、卷曲的膜组成。通过用针对E蛋白和Rab - 1（中间区室和内质网的标志物）的抗体进行共标记判断，E蛋白在这些高尔基体前体膜中积累并诱导其弯曲，此前已表明冠状病毒在此通过出芽进行组装。
