Dissecting Rotavirus Viroporin and Enterotoxin Calcium Signaling Pathways

儿童腹泻病是5岁以下儿童死亡的一个主要原因。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2.58亿例病例和20万人死亡。重要的是，RV研究长期以来一直用于建立对肠道病毒致病性的基本认识。因此，除了代表全球卫生负担之外，RV还提供了一个模型，我们可以从中深入了解其他病毒过程。RV感染的一个标志是Ca2+信号的失调，这是肠病毒发病和疾病的许多方面的基础。直到最近，这种失调被描述为细胞质Ca2+水平的单相增加。使用最先进的实时成像，我们发现RV感染导致不同的Ca2+信号事件，模仿以前表征的内源性信号。这些包括感染早期的细胞内Ca2+泡，以及后来的“细胞间Ca2+波”，涉及从rv感染到周围未感染细胞的旁分泌信号。RV非结构蛋白4 （NSP4）负责Ca2+稳态失调。通过不同的蛋白结构域，RV NSP4作为一种病毒孔蛋白（VD），引起Ca2+从内质网释放，并作为一种肠毒素（ED），引起受体依赖的瞬时Ca2+信号。虽然已知这些结构域参与Ca2+信号失调，但它们对复制和发病机制的相对贡献仍然不明确。本研究的总体目标是表征NSP4 VD和ED功能，描述这些结构域如何促进RV诱导的异常Ca2+信号，并确定Ca2+信号在RV复制和发病机制中的作用。在Aim 1中，我们将描述NSP4 VD和ED在RV诱导的Ca2+信号传导和RV复制中的作用。我们已经生成了一组具有靶向VD和ED突变的新型RV菌株。我们在RV反向遗传学，Ca2+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空白。此外，我们的团队首先确定了NSP4肠毒素和NSP4病毒蛋白的功能，因此可以适当地解决这些问题。我们预计大多数NSP4 VD突变体将减弱早期rv诱导的Ca2+信号和病毒复制。我们通常预计NSP4 ED的突变会减弱后来的Ca2+信号，特别是向邻近细胞的旁分泌信号。在Aim 2中，我们将确定NSP4 VD和ED对RV在人肠道和新生小鼠体内的体外致病性的贡献。我们预测，通过不同的功能，NSP4 VD和ED突变体将减轻RV腹泻和氯化物分泌，并将在不同方面促进致病性。总的来说，这些实验建立了关于病毒利用和模仿宿主信号和病理生理学机制的新概念，为提高疫苗效力提供了见解，并阐明了可能开发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.