p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly

ABSTRACTRespiratory syncytial virus (RSV) forms cytoplasmic inclusion bodies (IBs) that are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. The present study found that IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linkedN-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the posttranslational addition of OGN to protein targets to regulate cellular processes, including signal transduction, transcription, translation, and the stress response. The virus-induced sequestration of p38-P in IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: oxidative stress resulting from arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection.

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Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus

Nature Communications ◽

10.1038/s41467-017-00655-9 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 43

Author(s):

Vincent Rincheval ◽

Mickael Lelek ◽

Elyanne Gault ◽

Camille Bouillier ◽

Delphine Sitterlin ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Inclusion Bodies ◽

Functional Organization ◽

Cytoplasmic Inclusion ◽

Cytoplasmic Inclusion Bodies ◽

Syncytial Virus ◽

In Cells

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Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00013.2021 ◽

2021 ◽

Author(s):

Kempaiah Rayavara ◽

Alexander Kurosky ◽

Yashoda Madaiah Hosakote

Keyword(s):

Oxidative Stress ◽

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Stress Responses ◽

Transglutaminase 2 ◽

Lung Epithelial Cells ◽

Cross Linking ◽

Lung Epithelial ◽

Syncytial Virus ◽

Tract Infections

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways plays a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

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Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

mBio ◽

10.1128/mbio.01202-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 2

Author(s):

Marie Galloux ◽

Jennifer Risso-Ballester ◽

Charles-Adrien Richard ◽

Jenna Fix ◽

Marie-Anne Rameix-Welti ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Liquid Phase ◽

Inclusion Bodies ◽

Cytoplasmic Inclusion ◽

Cytoplasmic Inclusion Bodies ◽

Syncytial Virus ◽

P Proteins ◽

Nucleoprotein N ◽

Liquid Liquid Phase Separation

ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

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Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication

Journal of Virology ◽

10.1128/jvi.00260-10 ◽

2010 ◽

Vol 84 (23) ◽

pp. 12274-12284 ◽

Cited By ~ 101

Author(s):

Michael E. Lindquist ◽

Aaron W. Lifland ◽

Thomas J. Utley ◽

Philip J. Santangelo ◽

James E. Crowe

Keyword(s):

Respiratory Syncytial Virus ◽

Inclusion Bodies ◽

Binding Protein ◽

Mrna Translation ◽

Stress Granules ◽

Stress Granule ◽

Granule Formation ◽

Infected Cells ◽

Viral Inclusion ◽

Syncytial Virus

ABSTRACT Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.

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Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules

Journal of Virology ◽

10.1128/jvi.01168-19 ◽

2019 ◽

Vol 94 (3) ◽

Cited By ~ 4

Author(s):

Wanda Christ ◽

Janne Tynell ◽

Jonas Klingström

Keyword(s):

Protein Kinase ◽

Host Cell ◽

Stress Responses ◽

Stress Granules ◽

Cell Stress ◽

Stress Granule ◽

Hantavirus Infection ◽

Stress Signaling ◽

Protein Kinase R ◽

Granule Formation

ABSTRACT Virus infection frequently triggers host cell stress signaling resulting in translational arrest; as a consequence, many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study, we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs) but does so in only a limited proportion of infected cells. Our data indicate some cell type-specific and hantavirus species-specific variability in SG prevalence and show SG formation to be dependent on the activation of protein kinase R (PKR). Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like endoplasmic reticulum (ER) kinase (PERK). IMPORTANCE Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

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