Respiratory Syncytial Virus-Induced Oxidative Stress Leads to an Increase in Labile Zinc Pools in Lung Epithelial Cells

ABSTRACT Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection. IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance.

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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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Anti-inflammatory effect of MUC1 during respiratory syncytial virus infection of lung epithelial cells in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00225.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. L558-L563 ◽

Cited By ~ 46

Author(s):

Yusheng Li ◽

Darrell L. Dinwiddie ◽

Kevin S. Harrod ◽

Yong Jiang ◽

K. Chul Kim

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Inflammatory Responses ◽

Lung Epithelial Cells ◽

Apical Surface ◽

Lung Epithelial ◽

Rsv Infection ◽

Anti Inflammatory ◽

Syncytial Virus ◽

Inflammatory Effect

MUC1 is a transmembrane glycoprotein expressed on the apical surface of airway epithelial cells and plays an anti-inflammatory role during airway bacterial infection. In this study, we determined whether the anti-inflammatory effect of MUC1 is also operative during the respiratory syncytial virus (RSV) infection. The lung epithelial cell line A549 was treated with RSV, and the production of TNFα and the levels of MUC1 protein were monitored temporally during the course of infection by ELISA and Western blot analysis. Small inhibitory RNA (siRNA) transfection was utilized to assess the role of MUC1 in regulating RSV-mediated inflammatory responses by lung epithelial cells. Our results revealed that: 1) following RSV infection, an increase in MUC1 level was preceded by an increase in TNFα production and completely inhibited by soluble TNF receptor (TNFR); and 2) knockdown of MUC1 using MUC1 siRNA resulted in a greater increase in TNFα level following RSV infection compared with control siRNA treatment. We conclude that the RSV-induced increase in the TNFα levels upregulates MUC1 through its interaction with TNFR, which in turn suppresses further increase in TNFα by RSV, thus forming a negative feedback loop in the control of RSV-induced inflammation. This is the first demonstration showing that MUC1 can suppress the virus-induced inflammatory responses.

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