Sanderson C M, Parkinson J E, Hollinshead M, Smith G L
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
J Virol. 1996 Feb;70(2):905-14. doi: 10.1128/JVI.70.2.905-914.1996.
The vaccinia virus Western Reserve A38L protein is a hydrophobic integral membrane glycoprotein with amino acid similarity to mammalian integrin-associated protein. The protein has an N-terminal immunoglobulin superfamily domain, followed by five membrane-spanning domains and a short cytoplasmic tail. Deletion of the protein reduces virus plaque size but does not affect virus virulence (J. E. Parkinson, C. M. Sanderson, and G. L. Smith, Virology, in press). In this study, we have used a recombinant vaccinia virus in which the A38L gene may be inducibly overexpressed by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG), to demonstrate that overexpression of the vaccinia virus A38L gene produces drastic changes in the morphology, permeability, and adhesion of infected cells. In particular, A38L overexpression caused swelling of cells, marginalization of nuclear chromatin, and vacuolization of the endoplasmic reticulum, features characteristic of cell necrosis. By 18 h postinfection, cells become permeable and lytic as defined by the free entry of propidium iodide and loss of the cytoplasmic enzyme lactate dehydrogenase. Chelation of extracellular Ca2+ 22 h postinfection inhibited further release of lactate dehydrogenase, showing that Ca2+ influx was required for A38L-induced lysis. Direct measurement of 45Ca2+ influx showed that the rate of Ca2+ uptake was directly related to the period of A38L induction. The A38L protein, therefore, promotes the formation of pores within the plasma membrane of cells, and these pores facilitate Ca2+ entry and induce necrosis. Addition of rifampin inhibited virus assembly but not the ability of A38L to induce necrosis, indicating that pore formation is independent of viral morphogenesis. Finally, overexpression of the A38L protein resulted in a reduced plaque size and a threefold decrease in production of infective particles in vitro. The A38L protein represents the first example of a virus protein which directly or indirectly promotes the influx of extracellular Ca2+.
痘苗病毒西储株A38L蛋白是一种疏水性整合膜糖蛋白，与哺乳动物整合素相关蛋白在氨基酸序列上具有相似性。该蛋白具有一个N端免疫球蛋白超家族结构域，随后是五个跨膜结构域和一个短的胞质尾巴。缺失该蛋白会减小病毒蚀斑大小，但不影响病毒毒力（J. E. 帕金森、C. M. 桑德森和G. L. 史密斯，《病毒学》，即将发表）。在本研究中，我们使用了一种重组痘苗病毒，其中A38L基因可通过添加异丙基-β-D-硫代半乳糖苷（IPTG）进行诱导性过表达，以证明痘苗病毒A38L基因的过表达会使受感染细胞的形态、通透性和黏附性发生剧烈变化。特别是，A38L过表达导致细胞肿胀、核染色质边缘化以及内质网空泡化，这些都是细胞坏死的特征。感染后18小时，细胞变得具有通透性并发生裂解，这可通过碘化丙啶的自由进入和胞质酶乳酸脱氢酶的丧失来定义。感染后22小时螯合细胞外Ca2+可抑制乳酸脱氢酶的进一步释放，表明Ca2+内流是A38L诱导细胞裂解所必需的。对45Ca2+内流的直接测量表明，Ca2+摄取速率与A38L诱导的时间段直接相关。因此，A38L蛋白促进细胞质膜内孔的形成，这些孔有助于Ca2+进入并诱导坏死。添加利福平可抑制病毒组装，但不影响A38L诱导坏死的能力，这表明孔的形成与病毒形态发生无关。最后，A38L蛋白的过表达导致蚀斑大小减小，体外感染性颗粒产量降低三倍。A38L蛋白是病毒蛋白直接或间接促进细胞外Ca2+内流的首个实例。
