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

Alexandra L Chang-Graham,Alicia C Strtak,Asha A Philip,Jacob L Perry,Joseph M Hyser,John T Patton,Jeanette M Criglar,Mary K Estes,Nina K Ramachandran

doi:10.1038/s41598-019-46856-8

Alexandra L Chang-Graham, Alicia C Strtak + Show 7 more

Open Access

https://doi.org/10.1038/s41598-019-46856-8

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Abstract

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]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 [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]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 [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.

Highlights

RV was first reported to elevate cytosolic [Ca2+] by Michelangeli et al (1991), which stimulated subsequent research into how RV alters cellular Ca2+ levels[4]
We recently developed the use of genetically-encoded calcium indicators (GECIs) for the study of Ca2+ signaling in virus-infected cells[23]
We developed a series of cell lines stably expressing GECIs and used these cell lines to perform live-cell Ca2+ imaging over the course of a RV

Summary

Introduction

RV was first reported to elevate cytosolic [Ca2+] by Michelangeli et al (1991), which stimulated subsequent research into how RV alters cellular Ca2+ levels[4]. In RV-infected cells, ER-localized NSP4 is a viroporin (i.e., virus-encoded ion channel) that releases ER Ca2+ and reduces ER Ca2+ stores causing a persistent increase in cytosolic Ca2+ 6–8. Cell population-based studies show that RV induces a monophasic 2-fold increase in cytosolic Ca2+ during infection[18,19], but whether individual cells manifest this as a monophasic Ca2+ increase or a series of discrete Ca2+ signals remains unknown. The goal of this study was to use cell lines and human intestinal enteroids (HIEs) stably expressing cytoplasmic and/or ER-localized GECIs to define RV-induced Ca2+ signaling dynamics at the single-cell level, and thereby gain new mechanistic insights into how RV dysregulates Ca2+ homeostasis

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