Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca
            <sup>2+</sup>
            Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

Díaz, Yuleima; Chemello, María Elena; Peña, Franshelle; Aristimuño, Olga Carolina; Zambrano, José Luis; Rojas, Héctor; Bartoli, Fulvia; Salazar, L; Chwetzoff, Serge; Sapin, Catherine; Trugnan, Germain; Michelangeli, Fabián A.; Ruiz, Marie Christine

doi:10.1128/jvi.00577-08

Cited by 50 publications

(61 citation statements)

References 45 publications

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“…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 cells and a variety of mammalian cell lines, and NSP4 recapitulates all of the changes in Ca 2ϩ homeostasis observed in RV-infected cells (5,6). Because the NSP4-induced rapid and sustained increase in [Ca 2ϩ ]cyto is absolutely required for RV replication, several studies have sought to define the underlying mechanisms responsible for the elevation in [Ca 2ϩ ]cyto (4,5,7).…”

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confidence: 99%

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

Hyser

Utama

Crawford

et al. 2013

J Virol

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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...

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confidence: 99%

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

Hyser

Utama

Crawford

et al. 2013

J Virol

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“…The NSP4 (Wa strain, G1P1A [8]) recombinant gene cassette were confirmed by PCR, which produced approximately 750 bp sized amplicon (data not shown). DNA sequencing confirmed that the NSP4 recombinant gene cassette was fused in-frame into the plasmid pET23b due to the artificially incorporated restriction sites.…”

Section: Cloning and Expression Of Nsp4mentioning

confidence: 99%

“…NSP4 may function by causing Ca 2+ influx into the cytoplasm of infected cells (5,6). To study the biological properties of NSP4, the expression of large quantities of purified protein was studied in various eukaryotic and prokaryotic gene expression systems, including Escherichia coli (7,8).…”

Section: Introductionmentioning

confidence: 99%

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Role of Rotavirus Enterotoxin NSP4 in the Inflammatory Response in Murine Macrophage RAW 264.7 Cells

2016

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The rotavirus nonstructural glycoprotein, NSP4, has been identified as the first viral enterotoxin capable of inducing diarrhea. To investigate the biological function of NSP4 in the inflammatory process, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by RT-PCR, cloned into TA vector, and subsequently subcloned into pET23b expression plasmid. The expression of NSP4 protein was determined by SDS-PAGE and Western blotting, then, the protein was purified by affinity chromatography on Ni-NTA-agarose column. The inflammatory effects of NSP4, namely, production of nitric oxide (NO), pro-inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α), and prostaglandin E2 (PGE 2 ), was evaluated using NSP4-stimulated RAW 264.7 murine macrophages and compared with those observed after stimulation with lipopolysaccharide (LPS). The levels of IL-1β, IL-6, and TNF-α were significantly increased, and those of NO and PGE 2 also increased in NSP4-stimulated RAW 264.7 cells. These findings indicate that NSP4 plays an important role in the inflammatory response observed during rotavirus infection.

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“…NSP4, as an endoplasmic reticulum transmembrane glycoprotein, in association with VP7, stimulates viral parts construction and regulates viral morphogenesis . Moreover, NSP4, acting as a viral enterotoxin, provokes a significant increase in intracellular Ca 2+ volume concentration (Diaz et al, 2008), which is essential for stabilization of new virions VP7 protein; finally, it modifies integrity of intestinal epithelial cells, causing significant loss of water and electrolytes and, eventually, leading to the cellular necrosis (Tian et al, 1996;.…”

Section: Transmission Of the Virus And Patho-genesis Of The Infectionmentioning

confidence: 99%

Rotavirus infections in domestic animals

Chatzopoulos

Athanasiou

Spyrou

et al. 2017

J Hellenic Vet Med Soc

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ABSTRACT. Rotaviruses are major enteric pathogens of humans and a wide variety of animals. Rotavirus infections have a worldwide prevalence. The viral genome is composed of 11 double-stranded RNA segments with six structural and five or six non-structural proteins. Over 35,000 strains have been identified and classified into five main (A, B, C, D, E) and three additional tentative (F, G, H) serotype groups. A binary classification system has been proposed defining 'G' or 'P' types, with at least 27 G and 35 P genotypes reported thusfar. The virus is transmitted primarily by the faecal-oral and oral routes. After attachment, entry into the host cells occurs through direct entry, fusion or endocytosis. Main mechanisms of Rotavirus-induced diarrhoea involve extensive enterocyte losses and nutrient disdigestion and malabsorption. Clinical features of Rotavirus infections range from asymptomatic infections to fulminant disease leading to rapid death. In calves, lambs and kids, piglets and foals, salient sign of the disease is diarrhoea; diarrhoeic faeces are white, yellow or, in severe cases, blood-tinted or frank haemorrhagic. In dogs and cats, the infection occurs usually as self-limiting diarrhoea. Avian Rotavirus infections are characterized by enteritis, growth depression and/or growth retardation. Definitive diagnosis of the infection can only be achieved by laboratory tests, including electron microscopic examination, immunohistochemical examination, immunofluorescence, ELISA, latex agglutination and molecular techniques. There is no specific treatment against Rotavirus infections. Treatment is based in providing supportive care and managing clinical signs and potential complications. Effective vaccines, containing inactivated, recombinant or attenuated strains of the virus, are available. Challenge studies have shown the ease of the virus in cross-infecting various animal species; animal strains of the virus may cross species and infect humans. Due to the ability of the virus to overcome species barriers, animal strains may act as natural source of viral genomes, promoting mutations and creating new viral genotypes, whose virulence cannot be predictable.

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Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca ²⁺ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

Cited by 50 publications

References 45 publications

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

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

Role of Rotavirus Enterotoxin NSP4 in the Inflammatory Response in Murine Macrophage RAW 264.7 Cells

Rotavirus infections in domestic animals

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Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca 2+ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

Cited by 50 publications

References 45 publications

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

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

Role of Rotavirus Enterotoxin NSP4 in the Inflammatory Response in Murine Macrophage RAW 264.7 Cells

Rotavirus infections in domestic animals

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Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca ²⁺ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells