Dissecting Rotavirus Viroporin and Enterotoxin Calcium Signaling Pathways

儿科腹泻病是5岁以下儿童死亡的主要原因。轮状病毒(RV)是导致儿童腹泻疾病的主要原因，全世界每年估计有2.58亿例轮状病毒感染，20万人死亡。重要的是，轮状病毒研究长期以来一直被用来建立对肠道病毒致病性的基础了解。因此，除了代表全球健康负担之外，轮状病毒还提供了一个模型，我们可以从中洞察其他病毒过程。轮状病毒感染的一个特征是钙信号的失调，这是肠道病毒致病和疾病的许多方面的基础。直到最近，这种失调还被描述为胞浆钙水平的单相升高。使用最先进的实时成像技术，我们发现RV感染导致不同的钙信号事件，模仿先前特征的内源性信号。这些包括感染早期的细胞内钙离子喷发，以及后来的“细胞间钙波”，涉及从RV感染到周围未感染细胞的旁分泌信号。RV Non Structure Protein 4(NSP4)负责钙稳态的失调。RV NSP4通过不同的蛋白结构域发挥病毒孔蛋白(VD)和肠毒素(ED)的作用，前者引起内质网钙离子释放，后者引起受体依赖性的瞬时钙信号。虽然已知这些结构域参与了钙信号的失调，但它们在复制和发病机制中的相对作用仍不清楚。本研究的总体目标是确定NSP4的VD和ED功能，描述这些结构域如何在RV诱导的钙信号异常中起作用，并确定钙信号在RV复制和发病中的作用。在目标1中，我们将研究NSP4、VD和ED在RV诱导的钙信号转导和RV复制中的作用。我们已经产生了一组具有靶向VD和ED突变的新型RV毒株。我们在轮状病毒反向遗传学、钙信号和肠道生理学方面的丰富经验使我们非常适合研究这一知识差距。此外，我们的团队首先确定了nsp4肠毒素和nsp4病毒孔蛋白的功能，因此可以适当地解决这些问题。我们预计，大多数nsp4VD突变体将减弱RV诱导的早期钙信号和病毒复制。我们一般认为nsp4ED的突变将减弱后来的钙信号，特别是对邻近细胞的旁分泌信号。在目标2中，我们将确定NSP4、VD和ED对轮状病毒体外致病作用的影响，以及在新生小鼠体内致病作用。我们预测，通过不同的功能，NSP4VD和ED突变体将减轻轮状病毒的腹泻和氯离子分泌，并将在不同的致病性方面做出贡献。总的来说，这些实验建立了关于病毒利用机制以及宿主信号和病理生理学模拟的新概念，为提高疫苗效力提供了见解，并阐明了可能被开发为新的治疗靶点的靶点。

Pediatric diarrheal diseases are a major cause of mortality among children under 5 years old. Rotavirus (RV) is the leading cause of diarrheal diseases in children, with an estimated 258 million cases and 200,000 deaths each year worldwide. Importantly, RV studies have long been used to establish a foundational understanding of enteric virus pathogenicity. Thus, in addition to representing a global health burden, RV provides a model from which we can gain insight into other viral processes. A hallmark of RV infection is dysregulation of Ca2+ signaling which underlies many aspects of enteric virus pathogenesis and disease. Until recently, this dysregulation was described as a monophasic increase in cytosolic Ca2+ levels. Using state-of-the-art live imaging, we found that RV infection results in distinct Ca2+ signal events that mimic previously characterized endogenous signals. These include intracellular Ca2+ puffs early in infection, and later “intercellular Ca2+ waves” that involve paracrine signaling from RV-infected to surrounding uninfected cells. RV nonstructural protein 4 (NSP4) is responsible for dysregulating Ca2+ homeostasis. Through distinct protein domains, RV NSP4 functions as a viroporin (VD), causing Ca2+ release from the endoplasmic reticulum, and an enterotoxin (ED), which elicits a receptor- dependent transient Ca2+ signal. While these domains are known to be involved in Ca2+ signal dysregulation, their relative contributions to replication and pathogenesis remain poorly defined. The overall objective of this research is to characterize NSP4 VD and ED functions, delineate how these domains contribute to the aberrant RV-induced Ca2+ signals and determine the role Ca2+ signaling plays in RV replication and pathogenesis. In Aim 1 we will characterize the role of NSP4 VD and ED in RV-induced Ca2+ signaling and RV replication. We have generated a panel of novel RV strains with targeted VD and ED mutations. Our extensive experience with RV reverse genetics, Ca2+ signaling, and enteric physiology make us well-suited to investigate this gap-in-knowledge. Further, our team first identified both NSP4 enterotoxin and NSP4 viroporin functions and is thus aptly poised to address these questions. We anticipate that most NSP4 VD mutants will attenuate early RV-induced Ca2+ signaling and viral replication. We generally expect mutations in NSP4 ED will attenuate later Ca2+ signaling, specifically paracrine signaling to adjacent cells. In Aim 2 we will determine the contributions of NSP4 VD and ED to RV pathogenicity ex vivo in human intestinal enteroids and in vivo in neonatal mice. We predict that through distinct functions, NSP4 VD and ED mutants will attenuate RV diarrhea and chloride secretion and will contribute to different aspects of pathogenicity. Collectively, these experiments establish new concepts about mechanisms of viral exploitation and mimicry of host signaling and pathophysiology, provide insight for improved vaccine efficacy, and illuminate targets that might be exploited for novel therapeutic targets.