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: 10176473
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10176473
• 关键词: Address; Affect; Antidiarrheals; Antiviral Agents; 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; Structure; 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.

摘要 由感染、突变或药物引起的Ca 2+体内平衡紊乱可导致严重疾病。 了解调节健康和疾病的基本Ca 2+信号通路非常重要 促进医疗保健和药物开发。轮状病毒（RV）是研究如何 Ca 2+信号传导中断可能导致危及生命的疾病。RV是一种肠道病毒，可引起腹泻， 呕吐，这可能是致命的儿童没有支持性护理。RV感染高度局限于绒毛尖端 然而，这与广泛存在的肠道功能失调形成对比， 在感染期间。为了解决这个问题，RV发病机制的主要租户是病毒诱导的旁分泌信号传导， 感染的细胞转化为未感染的细胞RV引起广泛的Ca 2+信号失调，这是一个关键的介质 的发病机制内的RV复制和旁分泌信号通路的局部生态位。 已经做了很多工作来鉴定负责Ca 2+失调的病毒蛋白， 确定RV非结构蛋白4（NSP 4）为主要效应物。NSP 4宿主Ca 2+信号通过两个 不同的功能结构域，一个通过直接靶向在感染细胞内起作用[细胞内NSP 4（iNSP 4）] 宿主细胞的ER Ca 2+库，和从感染细胞分泌的第二种形式[细胞外NSP 4（eNSP 4）]， 在未感染的细胞中释放Ca 2+信号。iNSP 4和eNSP 4 Ca 2+信号都与细胞内Ca 2+的分子水平有关。 腹泻的决定因素，即肠神经系统的激活和Cl-分泌的过度激活。 然而，仍然存在重大的知识空白。首先，虽然NSP 4是Ca 2+的主要病毒效应子， 失调，它通过利用宿主Ca 2+信号传导途径来实现，但宿主蛋白和途径 特征不佳。第二，虽然由eNSP 4（和/或由宿主衍生的分子诱导的旁分泌信号传导， 通过RV）预测，没有直接证据表明RV感染的细胞间Ca 2+信号与RV感染的细胞间Ca 2+信号有关。 一个未被感染的细胞我们以前和新的初步工作表明，RV使用iNSP 4持续地 激活宿主的钙库操纵的钙进入（SOCE）途径。SOCE是一种重要的钙信号转导途径 在非兴奋性细胞中是一种有效的Cl-通路激活剂，通过Ca 2+激活的Cl-通道分泌Cl-。目的 1将研究RV如何利用SOCE，专注于细胞膜上称为ER-PM的微区 连接，其中SOCE Ca 2+进入发生和潜在耦合到Cl-分泌。此外，我们还生成了 新的细胞系和人类肠类，建立了第一个实验系统的直接，Ca 2 + 基于成像的可视化RV诱导的细胞间“Ca 2+波”，并确定这种旁分泌信号是 由未感染细胞上的嘌呤能受体介导。在目标2中，我们将研究分子机制 Ca 2+波的传播，并确定这一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。