Enteric Virus Exploitation of Store-operated and Purinergic Calcium Signaling

肠道病毒利用商店操作和嘌呤能钙信号传导

• 批准号: 10437691
• 负责人: Joseph M. Hyser
• 金额: $ 35.58万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-13 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10437691
• 关键词: Address; Affect; Antidiarrheals; Apical; Astrovirus; Biological Models; Calcium; Calcium Signaling; Caring; Cell Line; Cell membrane; Cell physiology; Cells; Child; Communication; Complex; Couples; Coupling; Cytoplasmic Tail; DNA Sequence Alteration; Data; Diarrhea; Disease; Endoplasmic Reticulum; Enteral; Enteric Nervous System; Enterochromaffin Cells; Enterocytes; Enterotoxins; Enterovirus; Fluids and Secretions; Goals; Health; Homeostasis; Human; Image; Infection; Intestines; Ion Channel; Knowledge; Life; Link; Liquid substance; Measures; Mediating; Mediator of activation protein; Medical; Molecular; Morbidity - disease rate; Mutation; Nonstructural Protein; Norovirus; Paracrine Communication; Pathogenesis; Pathway interactions; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Physiological; Proteins; Publishing; Purinoceptor; Recombinants; Role; Rotavirus; Rotavirus Infections; Serotonin; Signal Pathway; Signal Transduction; Small Intestines; Supportive care; Surface; System; Villus; Viral; Viral Proteins; Virus; Virus Replication; Visualization; Vomiting; Work; base; clinically relevant; density; diarrheal disease; drug development; extracellular; imaging modality; insight; interest; mortality; new therapeutic target; paracrine; pathogen; receptor; recruit; vaccine access

ABSTRACT Perturbations in Ca2+ homeostasis, caused by infections, mutations or drugs, can cause severe diseases. Understanding the fundamental Ca2+ signaling pathways that regulate both health and disease are important for advancing medical care and drug development. Rotavirus (RV) is an excellent model system to study how disrupted Ca2+ signaling can result in life-threatening illness. RV is an enteric virus that causes diarrhea and vomiting, which can be fatal in children without supportive care. RV infection is highly localized to the villus tips of the small intestine, and yet this is in contrast to the wide-spread dysregulation of intestinal functions caused during infection. To address this, a major tenant of RV pathogenesis is virus-induced paracrine signaling from infected cells to uninfected cells. RV causes a broad dysregulation in Ca2+ signaling and this is a key mediator of pathogenesis both within the localized niche of RV replication and for the paracrine signaling pathways. Much work has been done to identify the viral protein(s) responsible for the Ca2+ dysregulation, and have identified the RV nonstructural protein 4 (NSP4) as the major effector. NSP4 host Ca2+ signals through two distinct functional domains, one acting inside the infected cell [intracellular NSP4 (iNSP4)] by directly targeting the host cell's ER Ca2+ store, and a second form secreted from infected cells [extracellular NSP4 (eNSP4)] that elicits a Ca2+ signal in uninfected cells. Both iNSP4 and eNSP4 Ca2+ signals are associated with the molecular determinants of diarrhea, namely activation of the enteric nervous system and hyperactivation of Cl- secretion. However, important gaps-in-knowledge remain. First, while NSP4 is the main viral effector of the Ca2+ dysregulation, it does so by exploiting host Ca2+ signaling pathways, but the host proteins and pathways are poorly characterized. Second, while paracrine signaling by eNSP4 (and/or by host-derived molecules induced by RV) is predicted, there has been not directly evidence of an intercellular Ca2+ signal from a RV-infected to an uninfected cell. Our previous and new preliminary work has shown that RV uses iNSP4 to persistently activate the host's the store-operated Ca2+ entry (SOCE) pathway. SOCE is an important Ca2+ signaling pathway in non-excitable cells and a potent activator of Cl- secretion through Ca2+-activated Cl- channels. Aim 1 will investigate how RV exploits SOCE, focusing on microdomains at the cell membrane called ER-PM junctions, where SOCE Ca2+ entry occurs and potential coupling to Cl- secretion. Further, we have generated new cell lines and human intestinal enteroids to established the first experimental systems for direct, Ca2+ imaging-based visualization of RV-induced intercellular “Ca2+ waves” and identified that this paracrine signal is mediated by purinergic receptors on uninfected cells. In Aim 2, we will investigate the molecular mechanisms for the propagation of the Ca2+ wave and determine whether this purinergic signaling pathway is important for the activation of Cl- and serotonin secretion, and therefore a host mediator of RV diarrhea.

抽象的 由感染，突变或药物引起的Ca2+稳态的扰动可能引起严重的疾病。 了解调节健康和疾病的基本CA2+信号传导途径很重要 用于推进医疗和药物开发。轮状病毒（RV）是研究如何研究的绝佳模型系统 CA2+信号破坏会导致威胁生命的疾病。 RV是导致腹泻和 呕吐，在没有支持护理的情况下，可能是致命的。 RV感染高度位于绒毛技巧 小肠，但这与引起的肠功能的广泛失调相反 在感染期间。为了解决这个问题，RV发病机理的主要租户是病毒引起的旁分泌信号传导 感染细胞对未感染的细胞。 RV引起CA2+信号传导的广泛失调，这是关键的中介者 RV复制的局部利基和旁分泌信号通路内的发病机理。 已经做了很多工作来识别负责CA2+失调的病毒蛋白，并具有 将RV非结构蛋白4（NSP4）鉴定为主要效应子。 NSP4主机Ca2+信号通过两个 不同的功能域，一种作用在感染细胞内[细胞内NSP4（INSP4）]直接靶向 宿主细胞的ER Ca2+存储，第二种形式从受感染的细胞[细胞外NSP4（ENSP4）]中分泌 在未感染的细胞中引起Ca2+信号。 INSP4和ENSP4 Ca2+信号都与分子有关 腹泻的决定因素，即肠神经系统的激活和CL分泌过度激活。 但是，仍然存在重要的知识差距。首先，而NSP4是CA2+的主要病毒效应子 失调，它通过利用主机Ca2+信号通路来做到这一点，但是宿主蛋白和途径是 特征不佳。其次，而ENSP4的旁分泌信号传导（和/或由宿主衍生的分子诱导的 预测RV），没有直接证据表明来自RV感染的细胞间Ca2+信号 未感染的细胞。我们以前和新的初步工作表明，RV使用Insp4持续 激活主机的商店经营的Ca2+条目（SOCE）途径。 SOCE是一个重要的CA2+信号传导 通过Ca2+激活的Cl-通道的非驱散细胞中的途径和CL-分泌的潜在激活剂。目的 1将研究RV利用SOCE，重点是在称为ER-PM的细胞膜上 结，出现SOCE Ca2+进入的结合，并可能与CL分泌耦合。此外，我们已经产生了 新的细胞系和人肠肠todoids以建立了第一个直接CA2+的实验系统 基于成像的可视化RV诱导的细胞间“ Ca2+波”，并确定该旁分泌信号为 由未感染细胞上的嘌呤能受体介导。在AIM 2中，我们将研究分子机制 对于Ca2+波的传播，并确定此嘌呤能信号通路是否对 Cl-和5-羟色胺分泌的激活，因此是RV腹泻的宿主介质。