Role of Rotavirus NSP4 Viroporin and Enterotoxin Activity in Aberrant Calcium Signaling and Replication

摘要 轮状病毒(RV)是危及幼儿生命的腹泻疾病的主要原因，也是RV的一个标志。 感染是指宿主细胞内胞浆钙([Ca~(2+)]Cyto)升高。RV nsp4是一种多功能蛋白， 通过其病毒孔蛋白结构域(Vd)充当病毒离子通道(例如，病毒孔蛋白)以引起([Ca~(2+)]细胞)升高， 而肠毒素结构域(ED)可诱导受体介导的瞬时钙信号并引起腹泻。 老鼠。有趣的是，RV诱导的钙离子升高表现为一个复杂的动态信号机制，即 包括至少两种不同的钙信号类型：钙离子喷发和细胞间波(ICW)。这两个都是 宿主钙信号中已知的现象，但以前未发现与病毒诱导的钙有关 发信号。我们观察到，轮状病毒引起的钙离子喷发在感染早期以亚细胞、核周围的钙离子形式出现。 发布活动。后来在感染期间，在钙离子喷发开始后，轮状病毒感染的细胞触发了ICW，我们 以前的特征是通过感染细胞释放ADP，进而扩散和激活P2Y1 周围细胞上的嘌呤能受体。虽然这些异常的钙信号是由nsp4引起的，但相对的 NSP4Vd和ED对这些信号的贡献尚不清楚。首先，我建议利用反向遗传 重组轮状病毒NSP4Vd和Ed的构建及其关键氨基酸突变功能的研究 在本地病毒感染的情况下的残留。此外，nsp4中的这些中断将提供关键的见解 通过观察Ca~(2+)信号表型、生长发育等多个方面探讨病毒复制和致病机制 动力学、蛋白质生产、病毒质形成、氯离子分泌和5-羟色胺分泌。其次，使用活细胞 Ca~(2+)成像技术，我认为RV诱导的Ca~(2+)喷发来自RV NSP4 VD，而不是宿主 钙离子通道。使用nsp4VD突变体和实时共聚焦钙离子成像，我将表征RV钙离子泡芙， 并确定这些基本的钙释放事件是宿主还是NSP4产生的。这些信息 通过这一提议获得的信息将使我们能够获得关于病毒如何利用病毒钙离子的机械性见解 侵占宿主钙信号通路和重新编程细胞以促进病毒复制的通道。 此外，这些想法和实验将拓宽我们对其他钙离子传导方式的理解 病毒孔蛋白在感染过程中发挥作用，有助于发现其他干扰钙信号通路的病毒 用于病毒复制策略。总而言之，这些来自使用新房车的新想法和发现 反向遗传学平台和活细胞钙成像将开启一个研究推动进步的新纪元 分子病毒学和生理学领域。

ABSTRACT Rotavirus (RV) is the leading cause of life-threatening diarrheal disease in young children, and a hallmark of RV- infection is the elevation of cytosolic calcium ([Ca2+]cyto) in the host cell. RV NSP4 is a multifunctional protein that acts as a viral ion channel (e.g., viroporin) through its viroporin domain (VD) to cause an elevation in ([Ca2+]cyto), whereas the enterotoxin domain (ED) elicits a receptor-mediated transient Ca2+ signal and causes diarrhea in mice. Interestingly, the RV-induced elevation in Ca2+ manifests as a complex dynamic signaling regime that is comprised of at least two distinct Ca2+ signal types: Ca2+ puffs and intercellular waves (ICWs). Both of these are known phenomenon in host Ca2+ signaling but have not been previously associated with virus-induced Ca2+ signaling. We observed that RV-induced Ca2+ puffs occur early during infection as subcellular, perinuclear Ca2+ release events. Later during infection, after the onset of the Ca2+ puffs, RV-infected cells trigger ICWs, which we previously characterized are mediated by the infected cell releasing ADP that in turn diffuses and activates P2Y1 purinergic receptors on surrounding cells. While these aberrant Ca2+ signals are caused by NSP4, the relative contribution of the NSP4 VD and ED to these signals are not known. First, I propose to utilize reverse genetic engineering of RV NSP4 VD and ED to probe the function of this protein through mutagenesis of key amino acid residues in the context of a native viral infection. Furthermore, these disruptions in NSP4 will give key insights into multiple aspects of virus replication and pathogenesis by observing Ca2+ signaling phenotype, growth kinetics, protein production, viroplasm formation, Cl- secretion, and serotonin secretion. Secondly, using live cell Ca2+ imaging technology, I propose that RV-induced Ca2+ puffs are from the RV NSP4 VD and not from host Ca2+ channels. Using the NSP4 VD mutants and live confocal Ca2+ imaging, I will characterize the RV Ca2+ puffs, and determine whether these elementary Ca2+ release events are host or NSP4 generated. The information gained through this proposal will allow us to gain mechanistic insights about how viruses utilize viral Ca2+ channels to commandeer host Ca2+ signaling pathways and reprogram cells to facilitate viral replication. Furthermore, these ideas and experiments will broaden our understanding of how other Ca2+-conducting viroporins function during infections, aiding in the discovery of other viruses that disrupt Ca2+ signaling pathways for virus replication strategies. In conclusion, these new ideas and discoveries from the use of the new RV reverse genetics platform and live cell Ca2+ imaging will usher in a new era of research driving advancements in the field of molecular virology and physiology.