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

摘要 轮状病毒（RV）是幼儿中威胁生命的腹泻病的主要原因，并且是RV的标志- 感染是宿主细胞中胞质钙（[Ca 2 +]cyto）的升高。RV NSP 4是一种多功能蛋白， 充当病毒离子通道（例如，病毒孔蛋白）通过其病毒孔蛋白结构域（VD）引起（[Ca 2 +]cyto）升高， 而肠毒素结构域（艾德）释放受体介导的瞬时Ca 2+信号并导致腹泻， 小鼠有趣的是，RV诱导的Ca 2+升高表现为一种复杂的动态信号机制， 包括至少两种不同的Ca 2+信号类型：Ca 2+喷流和细胞间波（ICW）。这两个都是 在宿主Ca 2+信号传导中已知现象，但以前没有与病毒诱导的Ca 2+相关 发信号。我们观察到RV诱导的Ca 2+喷流在感染早期发生，作为亚细胞，核周Ca 2 + 释放事件。在感染后期，在Ca 2+喷流开始后，RV感染的细胞触发ICW， 先前表征的是由感染细胞释放ADP介导的，ADP反过来扩散并激活P2 Y1 周围细胞上的嘌呤受体。虽然这些异常的Ca 2+信号是由NSP 4引起的，但相对的Ca 2+信号是由NSP 4引起的。 NSP 4 VD和艾德对这些信号的贡献是未知的。首先，我建议利用反向遗传 通过关键氨基酸的突变，对RV NSP 4 VD和艾德进行工程改造，以探索该蛋白的功能 在天然病毒感染的情况下，此外，NSP 4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、 动力学、蛋白质产生、病毒质形成、Cl-分泌和5-羟色胺分泌。第二，使用活细胞 通过Ca 2+成像技术，我认为RV诱导的Ca 2+喷流来自RV NSP 4 VD，而不是来自宿主 Ca 2+通道使用NSP 4 VD突变体和活的共聚焦Ca 2+成像，我将表征RV Ca 2+喷流， 并确定这些基本Ca 2+释放事件是宿主还是NSP 4产生的。的信息 通过该提案获得的信息将使我们能够获得有关病毒如何利用病毒Ca 2+的机制见解 通道，以征用宿主Ca 2+信号通路和重编程细胞，以促进病毒复制。 此外，这些想法和实验将拓宽我们对其他Ca 2+传导机制的理解。 病毒孔蛋白在感染过程中发挥作用，有助于发现其他破坏Ca 2+信号通路的病毒 用于病毒复制策略。总之，这些新的想法和发现来自于新房车的使用 反向遗传学平台和活细胞Ca 2+成像将迎来一个新的研究时代， 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.