Tian P, Ball J M, Zeng C Q, Estes M K
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Virol. 1996 Oct;70(10):6973-81. doi: 10.1128/JVI.70.10.6973-6981.1996.
During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum (ER). As rotavirus particles mature, they lose their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell. The nonstructural glycoprotein NSP4 functions as an intracellular receptor in the ER membrane (K. S. Au, W. K. Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), and it has been hypothesized that NSP4 is involved in the removal of the envelope during viral morphogenesis (M. K. Estes and J. Cohen, Microbiol. Rev. 53:410-449, 1989; B. L. Petrie, M. K. Estes, and D. Y. Graham, J. Virol. 46:270-274, 1983). The purpose of the present study was to determine if NSP4 has a direct membrane destabilization activity (MDA) by using liposome leakage assays and electron microscopic visualization of liposome, microsome, and viral envelope disruption. The fluorescent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4. A region corresponding to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes. NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein. These results suggest that this region contains at least part of the functional domain of NSP4. Liposomes composed of phosphatidylcholine and microsomes (to simulate ER membranes) were broken when observed by electron microscopy after incubation with NSP4 or the NSP4(114-135) peptide. In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and erythrocytes were not disrupted by NSP4 and the NSP4(114-135) peptide. These results provide direct evidence that NSP4 possesses MDA and suggest that it can cause ER membrane damage. Therefore, NSP4 might play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. A model for the MDA of NSP4 in viral morphogenesis is proposed.
在一个独特的形态发生过程中，当新合成的亚病毒颗粒芽生进入内质网（ER）时，轮状病毒获得一层瞬时膜包膜。随着轮状病毒颗粒的成熟，它们失去其瞬时膜，糖蛋白VP7的一层形成病毒粒子的外衣壳。非结构糖蛋白NSP4在内质网膜中作为一种细胞内受体发挥作用（K. S. Au、W. K. Chan、J. W. Burns和M. K. Estes，《病毒学杂志》63:4553 - 4562，1989年），并且据推测NSP4参与病毒形态发生过程中包膜的去除（M. K. Estes和J. Cohen，《微生物学评论》53:410 - 449，1989年；B. L. Petrie、M. K. Estes和D. Y. Graham，《病毒学杂志》46:270 - 274，1983年）。本研究的目的是通过使用脂质体泄漏试验以及脂质体、微粒体和病毒包膜破坏的电子显微镜观察，来确定NSP4是否具有直接的膜去稳定化活性（MDA）。当脂质体与纯化的NSP4一起孵育时，掺入脂质体的荧光标记物（钙黄绿素）被释放出来。对应于NSP4氨基酸残基114至135的区域也能从脂质体中释放钙黄绿素。NSP4(114 - 135)肽特异性抗体完全阻断了纯化的NSP4蛋白的MDA。这些结果表明该区域至少包含NSP4功能域的一部分。与NSP4或NSP4(114 - 135)肽一起孵育后，通过电子显微镜观察发现，由磷脂酰胆碱组成的脂质体和微粒体（模拟内质网膜）被破坏。相比之下，源自细胞质膜的仙台病毒包膜和红细胞未被NSP4和NSP4(114 - 135)肽破坏。这些结果提供了直接证据表明NSP4具有MDA，并表明它可导致内质网膜损伤。因此，NSP4可能在病毒形态发生过程中从芽生颗粒上去除瞬时包膜方面发挥重要作用。提出了一个NSP4在病毒形态发生中的MDA模型。
