Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
mBio. 2010 Nov 30;1(5):e00265-10. doi: 10.1128/mBio.00265-10.
Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca(2+) ([Ca(2+)]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca(2+)]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca(2+) by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca(2+)]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca(2+)]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca(2+)]cyto, a critical event that regulates rotavirus replication and virion assembly.
许多病毒会改变细胞内钙离子稳态。轮状病毒非结构蛋白 4（NSP4）是一种内质网（ER）跨膜糖蛋白，通过一种与磷脂酶 C 无关的途径增加细胞质 Ca2+浓度 ([Ca2+]cyto)，这是病毒复制和形态发生所必需的。然而，增加 [Ca2+]cyto 的 NSP4 结构域和机制尚不清楚。我们鉴定了一个 NSP4 结构域（氨基酸 [aa] 47 至 90），该结构域插入膜中，具有 viroporin 的结构特征，viroporin 是一类小的疏水性病毒蛋白，它们破坏膜完整性和离子稳态，以促进病毒进入、组装或释放。突变分析表明，NSP4 viroporin 活性是由保守赖氨酸簇下游的一个两亲性 α 螺旋结构域介导的。赖氨酸簇指导 viroporin 结构域的完整膜插入，对于 viroporin 活性至关重要。在上皮细胞中，野生型 NSP4 的表达使游离细胞质 Ca2+水平增加了 3.7 倍，但 NSP4 viroporin 突变体保持低水平的 [Ca2+]cyto，保留在内质网中，并且不能形成细胞质囊泡结构，称为斑点，这些斑点围绕着轮状病毒感染细胞中的病毒复制和组装位点。当用 thapsigargin 药理学增加 [Ca2+]cyto 时，viroporin 突变体形成斑点，表明钙水平的升高和斑点的形成是 NSP4 的不同功能，表明 NSP4 直接或间接对细胞质钙水平的升高做出反应。NSP4 viroporin 活性建立了 NSP4 介导的 [Ca2+]cyto 升高的机制，这是调节轮状病毒复制和病毒粒子组装的关键事件。
