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      Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

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          Abstract

          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.

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          Most cited references44

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          STIM proteins: dynamic calcium signal transducers.

          Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca(2+)) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca(2+) stored within the ER lumen. As ER Ca(2+) is released to generate primary Ca(2+) signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca(2+)-selective Orai channels to mediate finely controlled Ca(2+) signals and to homeostatically balance cellular Ca(2+). Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.
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            Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.

            The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host-microbial interactions).
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              Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology.

              Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion.
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                Author and article information

                Contributors
                Joseph.Hyser@bcm.edu
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                25 July 2019
                25 July 2019
                2019
                : 9
                : 10822
                Affiliations
                [1 ]Alkek Center for Metagenomic and Microbiome Research, Department of Molecular Virology and Microbiology, Houston, USA
                [2 ]ISNI 0000 0001 2160 926X, GRID grid.39382.33, Department of Medicine, Gastroenterology and Hepatology and Infectious Disease, , Baylor College of Medicine, ; Houston, TX 77303 USA
                [3 ]ISNI 0000 0001 0790 959X, GRID grid.411377.7, Department of Biology, , Indiana University, ; Bloomington, IN 47405 USA
                Author information
                http://orcid.org/0000-0003-1572-0732
                Article
                46856
                10.1038/s41598-019-46856-8
                6658527
                31346185
                685b5a5c-41ac-4b46-b55b-8a1cb5f2f81f
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 May 2019
                : 4 July 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000002, U.S. Department of Health & Human Services | National Institutes of Health (NIH);
                Award ID: T32GM008231
                Award ID: T32GM008231
                Award ID: T32GM008231
                Award ID: R01AI080656
                Award ID: U19AI116497
                Award ID: K01DK093657
                Award ID: R03DK110270
                Award ID: R01DK115507
                Award Recipient :
                Categories
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                © The Author(s) 2019

                Uncategorized
                rotavirus,virus-host interactions
                Uncategorized
                rotavirus, virus-host interactions

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