Dissecting Rotavirus Viroporin and Enterotoxin Calcium Signaling Pathways

剖析轮状病毒病毒孔蛋白和肠毒素钙信号通路

基本信息

批准号：
10677701
负责人：
Sue Ellen Crawford
金额：
$ 40万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-21 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10677701
关键词：
5 year old Acute Address Agonist Antidiarrheals Astrovirus Attenuated Biological Models Calcium Signaling Cell Communication Cell membrane Cells Cessation of life Child Childhood Chlorides Communication Cytoplasmic Tail Cytosol Defect Diarrhea Disease Disease Pathway Drug Targeting Dysentery Endoplasmic Reticulum Enteral Enterocytes Enterotoxins Event Exhibits Functional disorder Genetic Goals Homeostasis Hour Human Image Infection Intestines Ion Channel Knowledge Maps Mediating Modeling Molecular Mus Mutation Nonstructural Protein Norovirus Nuclear Paracrine Communication Pathogenesis Pathogenicity Pathway interactions Physiology Play Point Mutation Process Protein Biosynthesis Proteins Recombinant Proteins Research Role Rotavirus Rotavirus Infections Rotavirus Vaccines Signal Pathway Signal Transduction System Tertiary Protein Structure Vaccines Viral Viral Pathogenesis Virus Virus Diseases Virus Replication Vomiting diarrheal disease experience experimental study extracellular fascinate global health human pathogen improved in vivo insight knock-down mimicry mortality mutant neonatal mice new therapeutic target novel pathogen pathogenic virus programs protein expression pup receptor reverse genetics theories tool vaccine access vaccine efficacy

项目摘要

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.

小儿腹泻疾病是5岁以下儿童死亡的主要原因。轮状病毒（RV）是儿童腹泻疾病的主要原因，全球估计有2.58亿例和20万例死亡。重要的是，长期以来，RV研究已被用来建立对肠道病毒致病性的基础理解。除了代表全球健康伯恩外，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感染的标志是CA2+信号传导失调，其基础是肠道病毒发病机理和疾病的许多方面。直到最近，这种失调被描述为胞质Ca2+水平的单相增加。使用最新的实时成像，我们发现RV感染会导致不同的CA2+信号事件，这些事件模仿了内源性信号。这些包括感染早期的细胞内Ca2+泡芙，后来包括涉及从感染的RV感染到周围未感染细胞的旁分泌信号传导的“细胞间CA2+波。 RV非结构蛋白4（NSP4）导致CA2+稳态失调。通过不同的蛋白质结构域，RV NSP4充当Viroporin（VD），导致内质网的Ca2+释放，而肠毒素（ED）引起了接收器依赖性瞬态CA2+信号。尽管已知这些结构域参与Ca2+信号失调，但它们对复制的相对贡献和发病机理的定义仍然很差。这项研究的总体目的是表征NSP4 VD和ED功能，描述这些域如何促进异常RV诱导的Ca2+信号，并确定CA2+信号传导在RV复制和发病机理中的作用。在AIM 1中，我们将表征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+信号传导和肠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病毒孢子蛋白功能，因此恰当地毒害了我们预计大多数NSP4 VD突变体将减轻早期RV诱导的Ca2+信号传导和病毒复制。我们通常预计NSP4 ED中的突变会减弱以后的Ca2+信号传导，特别是旁分泌信号传导到相邻细胞。在AIM 2中，我们将确定NSP4 VD和ED对人体肠内型和新生儿小鼠体内体内RV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，并有助于致病性的不同方面。总的来说，这些实验建立了有关病毒剥削机制和宿主信号传导和病理生理学的模仿机制的新概念，为提高疫苗效率提供了见识，并阐明了可能被利用的靶标，这些靶标可能会用于新的治疗靶点。