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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...
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...
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 ...
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