Enteric Virus Exploitation of Store-operated and Purinergic Calcium Signaling

摘要 感染、突变或药物引起的钙稳态紊乱可能会导致严重的疾病。 了解调节健康和疾病的基本钙信号通路是很重要的 用于促进医疗保健和药物开发。轮状病毒(RV)是研究轮状病毒 钙信号受阻会导致危及生命的疾病。轮状病毒是一种肠道病毒，会导致腹泻和 呕吐，这对没有支持性护理的儿童来说可能是致命的。轮状病毒感染高度局限于绒毛尖端 但这与广泛存在的肠道功能失调形成了鲜明对比 在感染期间。为了解决这个问题，轮状病毒致病机制的一个主要租户是病毒诱导的旁分泌信号。 受感染的细胞到未受感染的细胞。RV引起钙信号的广泛失调，这是一个关键的调节因子 在RV复制的局部生态位内和旁分泌信号通路中都存在致病机制。 人们已经做了很多工作来确定导致钙离子失调的病毒蛋白(S)，并已经 确定RV非结构蛋白4(NSP4)为主要效应因子。NSP4通过两个 不同的功能区，通过直接靶向作用于受感染的细胞内[细胞内nsp4(INSP4)] 宿主细胞的内质网钙离子储存，感染细胞分泌的第二种形式[细胞外nsp4(ENSP4)] 在未感染的细胞中引发钙信号。INSP4和eNSP4钙信号都与分子相关。 腹泻的决定因素，即肠道神经系统的激活和氯离子分泌的过度激活。 然而，重要的知识差距仍然存在。首先，虽然NSP4是钙离子的主要病毒效应因子 失调，它通过利用宿主钙信号通路来做到这一点，但宿主蛋白和通路是 特征不佳的。第二，eNSP4(和/或由宿主衍生的分子诱导)的旁分泌信号 通过轮状病毒)被预测，目前还没有直接证据表明细胞内钙信号来自轮状病毒感染到 一个未受感染的细胞。我们以前和新的初步工作表明，RV使用iNSP4来持久地 激活宿主的存储操作的钙内流(SOCE)途径。SOCE是一种重要的钙信号转导途径 在不可兴奋的细胞中的途径，以及通过钙激活的氯离子通道分泌氯离子的有效激活剂。目标 1将研究RV如何利用SOCE，重点是细胞膜上称为ER-PM的微域 连接，其中SOCE钙离子进入和潜在的耦合到氯-分泌。此外，我们还产生了 新的细胞系和人类肠道肠样建立了第一个直接、钙离子的实验系统 基于成像的可视化RV诱导的细胞间“钙波”，并确认这个旁分泌信号是 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。 并确定这条嘌呤能信号通路是否对 激活氯离子和5-羟色胺的分泌，因此是轮状病毒腹泻的宿主介质。

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.