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 ...