Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca 2+ ]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca 2+ ]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca 2+ ]cyt signaling that manifest as hundreds of discrete [Ca 2+ ]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca 2+ ]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca 2+ ]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca 2+ ]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca 2+ ]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca 2+ ]cyt by dynamic calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5′-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Enteric viruses in the Caliciviridae family cause acute gastroenteritis in humans and animals, but the cellular processes needed for virus replication and disease remain unknown. A common strategy among enteric viruses, including rotaviruses and enteroviruses, is to encode a viral ion channel (i.e., viroporin) that is targeted to the endoplasmic reticulum (ER) and disrupts host calcium (Ca2+) homeostasis. Previous reports have demonstrated genetic and functional similarities between the nonstructural proteins of caliciviruses and enteroviruses, including the calicivirus NS1-2 protein and the 2B viroporin of enteroviruses. However, it is unknown whether caliciviruses alter Ca2+ homeostasis for virus replication or whether the NS1-2 protein has viroporin activity like its enterovirus counterpart. To address these questions, we used Tulane virus (TV), a rhesus enteric calicivirus, to examine Ca2+ signaling during infection and determine whether NS1-2 has viroporin activity that disrupts Ca2+ homeostasis. We found that TV increases Ca2+ signaling during infection and that increased cytoplasmic Ca2+ levels are important for efficient replication. Further, TV NS1-2 localizes to the endoplasmic reticulum, the predominant intracellular Ca2+ store, and the NS2 region has characteristics of a viroporin domain (VPD). NS1-2 had viroporin activity in a classic bacterial functional assay and caused aberrant Ca2+ signaling when expressed in mammalian cells, but truncation of the VPD abrogated these activities. Together, our data provide new mechanistic insights into the function of the NS2 region of NS1-2 and support the premise that enteric viruses, including those within Caliciviridae, exploit host Ca2+ signaling to facilitate their replication. IMPORTANCE Tulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal disease. Globally, enteric caliciviruses affect both humans and animals and amass >65 billion dollars per year in treatment and health care-associated costs, thus imposing an enormous economic burden. Recent progress has resulted in several cultivation systems (B cells, enteroids, and zebrafish larvae) to study human noroviruses, but mechanistic insights into the viral factors and host pathways important for enteric calicivirus replication and infection are still largely lacking. Here, we used Tulane virus, a calicivirus that is biologically similar to human noroviruses and can be cultivated by conventional cell culture, to identify and functionally validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses provides a novel approach to developing antivirals and comprehensive therapeutics for enteric calicivirus diarrheal disease outbreaks.

27Enteric viruses in the Caliciviridae family cause acute gastroenteritis in humans and animals, but 28 the cellular processes needed for virus replication and disease remain unknown. A common 29 strategy among enteric viruses, including rotaviruses and enteroviruses, is to encode a viral ion 30 channel (i.e., viroporin) that is targeted to the endoplasmic reticulum (ER) and disrupts host 31 calcium (Ca 2+ ) homeostasis. Previous reports have demonstrated genetic and functional 32 similarities between the nonstructural proteins of caliciviruses and enteroviruses, including the 33 calicivirus NS1-2 protein and the 2B viroporin of enteroviruses. However, it is unknown whether 34 caliciviruses alter Ca 2+ homeostasis for virus replication or whether the NS1-2 protein has 35 viroporin activity like its enterovirus counterpart. To address these questions, we used Tulane 36 virus (TV), a rhesus enteric calicivirus, to examine Ca 2+ signaling during infection and determine 37 whether NS1-2 has viroporin activity that disrupts Ca 2+ homeostasis. We found that TV disrupts 38 increases Ca 2+ signaling during infection and increased cytoplasmic Ca 2+ levels is important for 39 efficient replication. Further, TV NS1-2 localizes to the endoplasmic reticulum (ER), the 40 predominant intracellular Ca 2+ store and the NS2 region has characteristics of a viroporin domain 41 (VPD). NS1-2 had viroporin activity in a classic bacterial functional assay and caused aberrant 42 Ca 2+ signaling when expressed in mammalian cells, but truncation of the VPD abrogated these 43 functions. Together, our data provide new mechanistic insights into the function of the NS2 44 region of NS1-2 and show that like many other enteric viruses, enteric caliciviruses also exploit 45 host Ca 2+ signaling to facilitate their replication. 46 Importance 47Tulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal 48 disease. Globally, enteric caliciviruses affect both humans and animals and result in >65 billion 49 dollars per year in treatment and healthcare-associated costs, thus imposing an enormous 50 economic burden. Recent progress has resulted in several cultivation systems (B cell, enteroid 51 and zebrafish larvae) to study human noroviruses, but mechanistic insights into the viral factors 52 and host pathways important for enteric calicivirus replication and infection are largely still 53 lacking. Here we used Tulane virus, a calicivirus that is biologically similar to human 54 noroviruses and can be cultivated in conventional cell culture, to identify and functionally 55 validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be 56 a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses 57 provides a novel approach to developing antivirals and comprehensive therapeutics for enteric 58 calicivirus diarrheal disease outbreaks. 59 60 61The Caliciviridae family consists of small, non-enveloped single-stranded RNA viruses 62 with five ...

26Rotavirus causes severe diarrheal disease in children worldwide. A hallmark of rotavirus 27 infection is an increase in cytosolic calcium in infected small intestine epithelial cells. However, 28 the underlying mechanism(s) of rotavirus-cell signaling remains incompletely characterized. Here 29 we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular 30 calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids 31 (HIEs). Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) 32 that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 33 antagonists or CRISPR/Cas9 knockout of P2Y1. This paracrine purinergic signal is critical for 34 rotavirus replication and diarrhea. Blocking the ICW signal reduces rotavirus replication; inhibits 35 rotavirus-induced serotonin release and fluid secretion; and reduces diarrhea severity in neonatal 36 mice. This is the first evidence that viruses exploit intercellular calcium waves to amplify diarrheal 37 signaling; a finding which have broad implications for gastrointestinal physiology. signaling molecules during RV infection, including enterotoxin NSP4, prostaglandins (PGE2), and 57 nitric oxide (NO) 7,15-17 . In this model, enterotoxin NSP4 can bind to neighboring, uninfected 58 enterocytes to activate Ca 2+ -activated chloride channels and cause secretory diarrhea 18,19 , and 59 PGE2 and NO may further activate fluid secretion processes 20,21 . Dysregulation of neighboring 60 enteroendocrine cells triggers the Ca 2+ -dependent release of serotonin, which stimulates the 61 enteric nervous system both to activate vomiting centers in the central nervous system and to 62 activate secretory reflex pathways in the gastrointestinal (GI) tract 5,22 . Thus, this model of RV-63 induced diarrhea addresses how limited infection at the middle-to-upper villi may cause 64 widespread dysregulation of host physiology and life-threatening disease. 65Herein we demonstrate that RV-infected cells signal to uninfected cells via an extracellular 66 purinergic signaling pathway. This newly identified pathway is a dominant driver of observed RV 67 disease processes, including replication, upregulation of PGE2-and NO-producing enzymes, 68 serotonin secretion, fluid secretion, and diarrhea in a neonatal mouse model. Our findings provide 69 new insights into the mechanism(s) of viral diarrhea and gastrointestinal physiology. 70 Results 71 Low multiplicity infection reveals intercellular calcium waves 72Previous studies have shown that RV significantly increases cytosolic Ca 2+ during infection 73 and disrupts host Ca 2+ -dependent processes to cause disease [23][24][25] . We used African Green 74 monkey kidney MA104 cells stably expressing the genetically-encoded calcium indicator (GECI) 75GCaMP5G or GCaMP6s to observe changes in cytosolic Ca 2+ during RV infection using live-cell 76 time-lapse epifluorescence imaging. We did not observe differences in response using either...

28 Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating 29 cytosolic calcium ([Ca 2+ ]cyt) and decreasing endoplasmic reticulum (ER) stores. While 30 an overall, monophasic increase in [Ca 2+ ]cyt during RV infection has been shown, the 31 nature of the RV-induced aberrant calcium signals and how they manifest over time at 32 the single-cell level have not been characterized. Thus, we generated cell lines and 33 human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted 34 genetically-encoded calcium indicators to characterize calcium signaling throughout RV 35 infection by time-lapse imaging. We found that RV induces highly dynamic [Ca 2+ ]cyt 36 signaling that manifest as hundreds of discrete [Ca 2+ ]cyt spikes, which increase during 37 peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca 2+ ]cyt 38 spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced 39 [Ca 2+ ]cyt spikes were primarily from ER calcium release and were attenuated by 40 inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs 41 also exhibited prominent [Ca 2+ ]cyt spikes that were attenuated by inhibiting SOCE, 42 underlining the relevance of these [Ca 2+ ]cyt spikes to gastrointestinal physiology and 43 role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca 2+ ]cyt 44 by dynamic Ca 2+ signaling, establishes a new, paradigm-shifting understanding of the 45 spatial and temporal complexity of virus-induced Ca 2+ signaling. 46 47 48 Eukaryotic signal transduction pathways employ a variety of signaling molecules 49 to regulate cellular processes. Calcium (Ca 2+ ) is one of the most ubiquitous secondary 50 messengers in the cell, which tightly regulates Ca 2+ movement through the coordinated 51 function of Ca 2+ channels, transporters, and pumps. Since Ca 2+ signaling modulates a 52 wide array of cellular processes, it is not surprising that many different viruses exploit 53 Ca 2+ signaling to facilitate their replication, and the resulting dysregulation of Ca 2+ 54 signaling causes pathogenesis. Rotavirus (RV), a member of the Reoviridae family, is 55 one of the first viruses shown to elevate cellular Ca 2+ levels and has become a widely-56 used model system to characterize mechanisms by which viruses dysregulate host Ca 2+ 57 homeostasis 1 . RV is a clinically important enteric virus that causes severe diarrhea and 58 vomiting in children, resulting in over approximately 258 million diarrhea episodes and 59 198,000 deaths in 2016 2 . Hyperactivation of cyclic nucleotide (e.g., cAMP/cGMP) and 60 Ca 2+ signaling pathways is a common strategy among enteric pathogens 3 . Thus, 61 understanding how RV exploits Ca 2+ signaling is key to understanding and combating 62 RV-induced diarrhea. 63 RV was first reported to elevate cytosolic [Ca 2+ ] by Michelangeli et al. (1991), 64 which stimulated subsequent research into how RV alters cellular Ca 2+ levels 4 . RV 65 causes a 2-fold steady-state increase in ...

Despite causing severe, potentially life‐threatening diarrhea, most enteric viruses only infect a small percent of intestinal epithelial cells. Thus, the induction of paracrine signaling pathways to dysregulate both infected and neighboring uninfected cell remains a major premise of enteric virus pathogenesis. However, virus‐induced paracrine signaling has never been directly observed and therefore the mechanisms have yet to be defined. Rotavirus (RV) remains a major cause of severe diarrhea in children worldwide. A hallmark of RV infection is the activation of aberrant calcium (Ca2+) signaling, which is necessary for replication and activation of secretory pathways in GI epithelium. We sought to characterize RV‐induced calcium signaling dynamics and identify paracrine signaling pathways responsible for infected‐to‐uninfected cell signaling. We conducted live‐cell Ca2+ imaging throughout the entire infection using cell lines and human intestinal enteroids (HIEs) engineered to stably express the genetically‐encoded Ca2+ indicator GCaMP. We found that rotavirus significantly increases both steady‐state and transient Ca2+ signaling mediated by RV nonstructural protein 4 (NSP4) and mutation of the NSP4 ion channel domain altered the RV‐induced Ca2+ signaling pattern observed, particularly low amplitude Ca2+ puffs observed early in infected cells. Further, isolated rotavirus‐infected cells generated multiple intercellular calcium waves (ICWs), which was the most prominent paracrine signal observed in both MA104 cells and HIEs. RV infection induces several signaling molecules, such as enterotoxin NSP4, prostaglandins and nitric oxide, and in many cell types extracellular purinergic signaling by ATP/ADP are responsible for ICW propagation. We found that RV‐induced ICWs were abolished by blocking extracellular ATP/ADP signaling with apyrase or purinergic receptor inhibitors or by CRISPR/Cas9‐mediated deletion of the P2Y1 receptor, but not by blocking extracellular enterotoxin form of NSP4, nitric oxide, or prostaglandin signaling. Rotavirus‐induced paracrine signaling was critical for multiple aspects of rotavirus pathogenesis, including fluid secretion and serotonin release, as well as regulating host responses, such as upregulation of IL‐1α and mucin secretion. Finally, we investigated whether activation of aberrant Ca2+ signaling and paracrine purinergic signaling were features of enteric caliciviruses using Tulane virus (TV), a rhesus monkey calicivirus. Like RV, we found that TV encodes for a viral ion channel in the endoplasmic reticulum and induces aberrant Ca2+ signaling during infection. TV infected cells also trigger ICWs that are blocked by purinergic signaling inhibitors. Thus, enteric viruses employ mechanisms to not only disrupt signaling in the virus‐infected cell but also exploit paracrine purinergic signaling to generate ICWs that represent a potent mechanism to amplify the pathophysiological signals underlying viral diarrhea.