Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Abstract

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2-to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PMlocalized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections. C alcium (Ca 2ϩ ) is a ubiquitous secondary messenger, and the concentration of intracellular Ca 2ϩ is tightly regulated. As obligate intracellular parasites, viruses subvert host cell pathways to support robust virus replication. Many viruses disrupt host Ca 2ϩ homeostasis in order to establish a cellular environment conducive for virus replication and assembly (1). One well-established hallmark of rotavirus (RV) infection is dramatic changes in cellular Ca 2ϩ homeostasis, including increased permeability of the endoplasmic reticulum (ER), resulting in decreased ER Ca 2ϩ stores and activation of Ca 2ϩ influx channels in the plasma membrane (PM), ultimately resulting in an elevated cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ]cyto) (2-4). While both ER Ca 2ϩ stores and extracellular Ca 2ϩ contribute to the increased [Ca 2ϩ ]cyto, the extracellular pool is much greater than the ER stores; therefore, Ca 2ϩ influx through the PM likely accounts for the bulk of the increase in [Ca 2ϩ ]cyto in RV-infected cells. Using expression of individual recombinant RV proteins, nonstructural protein 4 (NSP4) was identified as the sole RV protein responsible for the elevation in [Ca 2ϩ ]cyto levels in Sf9 insect...

Modulation of Autophagy by Calcium Signalosome in Human Disease

“…Basically, the viral ER-anchored glycoprotein NSP4, a pore-forming viroporin, elicits the leakage of Ca 21 from the ER to the cytosol. This ion mobilization, in turn, activates autophagy through the CaMKK2-AMPK pathway; CaMKK2 inhibition by (Hyser et al, 2013). Later, the rotavirus also interferes with autophagy membrane trafficking to transport viral ER-associated proteins to sites of genome replication and particle assembly since lower expression of ATG3 and ATG5 highly reduce virus yield.…”

Ebola Virus Delta Peptide Is a Viroporin

“…Well-described viroporins are known from influenza virus, HIV, picornaviruses, rotaviruses, coronaviruses, and others, while putative viroporins have been reported for many other viruses (18,19,(38)(39)(40). Viroporins can act intracellularly to permeabilize organelle membranes (41), modulate calcium homeostasis (42), alter organelle acidification (43), induce autophagy (44), and otherwise "customize host cells for efficient viral propagation" (41). Other viroporins may act extracellularly and permeabilize infected or noninfected cells (18).…”

The effect of bovine rotavirus and its nonstructural protein 4 on ER stress-mediated apoptosis in HeLa and HT-29 cells

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