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：
10372424
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/10372424
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 Foundations Functional disorder Gap Junctions Genetic Goals Homeostasis Hour Human Image Infection Intestines Ion Channel Knowledge Maps Mediating Modeling Molecular Mus Mutation Nonstructural Protein Norovirus 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 protein expression pup receptor reverse genetics 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，全球每年估计有 2.58 亿病例和 20 万人死亡。因此，除了代表全球健康负担外，RV 还提供了一个模型，使我们能够深入了解 RV 感染的其他病毒过程。 Ca2+ 信号传导失调是肠道病毒发病机制和疾病的许多方面的基础，直到最近，这种失调被描述为胞质 Ca2+ 水平的单相增加，使用最先进的实时成像，我们发现 RV 感染会导致明显的后果。模仿先前表征的内源信号的 Ca2+ 信号事件包括感染早期的细胞内 Ca2+ 喷发，以及后来的“细胞间 Ca2+ 波”，涉及从 RV 感染到周围未感染的旁分泌信号传导。 RV 非结构蛋白 4 (NSP4) 负责调节 Ca2+ 稳态，RV NSP4 充当病毒孔蛋白 (VD)，导致 Ca2+ 从内质网释放，并产生肠毒素 (ED)，从而引发受体。 - 依赖性瞬时 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不清楚。这项研究的目的是表征 NSP4 VD 和 ED 功能，描述这些结构域如何促进异常 RV 诱导的 Ca2+ 信号，并确定 Ca2+ 信号在 RV 复制和发病机制中的作用。在目标 1 中，我们将表征 NSP4 VD 和 ED 的作用。 RV 诱导的 Ca2+ 信号传导和 RV 复制中的 ED 我们在 RV 反向遗传学、Ca2+ 信号传导和肠道方面拥有丰富的经验，已培育出一系列具有靶向 VD 和 ED 突变的新型 RV 菌株。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，因此我们预计大多数 NSP4 VD 突变体将减弱早期 RV- 的作用。我们通常预计 NSP4 ED 的突变会减弱随后的 Ca2+ 信号传导，特别是在目标 2 中，我们将减弱对邻近细胞的旁分泌信号传导。确定 NSP4 VD 和 ED 对 RV 体外致病性和新生小鼠体内的 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，并将有助于不同方面的 RV 腹泻和氯离子分泌。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传导和病理生理学模拟的新概念，为提高疫苗功效提供了见解，并阐明了可能用于新型治疗的靶点。目标。