Respiratory Viral Infections and Subversion of Cellular Antioxidant Defenses

Abstract

Reactive oxygen species (ROS) formation is part of normal cellular aerobic metabolism, due to respiration and oxidation of nutrients in order to generate energy. Low levels of ROS are involved in cellular signaling and are well controlled by the cellular antioxidant defense system. Elevated levels of ROS generation due to pollutants, toxins and radiation exposure, as well as infections, are associated with oxidative stress causing cellular damage. Several respiratory viruses, including respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza, induce increased ROS formation, both intracellularly and as a result of increased inflammatory cell recruitment at the site of infection. They also reduce antioxidant enzyme (AOE) levels and/or activity, leading to unbalanced oxidative-antioxidant status and subsequent oxidative cell damage. Expression of several AOE is controlled by the activation of the nuclear transcription factor NF-E2-related factor 2 (Nrf2), through binding to the antioxidant responsive element (ARE) present in the AOE gene promoters. While exposure to several pro-oxidant stimuli usually leads to Nrf2 activation and upregulation of AOE expression, respiratory viral infections are associated with inhibition of AOE expression/activity, which in the case of RSV and hMPV is associated with reduced Nrf2 nuclear localization, decreased cellular levels and reduced ARE-dependent gene transcription. Therefore, administration of antioxidant mimetics or Nrf2 inducers represents potential viable therapeutic approaches to viral-induced diseases, such as respiratory infections and other infections associated with decreased cellular antioxidant capacity.

Keywords: Free radicals; Nrf2; Oxidative stress; ROS; Respiratory syncytial virus.

Figure 1. RSV and hMPV infection modulates ARE-dependent gene transcription

(A) A549 cells were transiently transfected with a plasmid containing multiple copies of the NQO1 ARE site linked to the luciferase gene and then infected with either RSV (Left panel) or hMPV (Right panel). Cells were harvested at different times post-infection to measure luciferase activity. Uninfected cells, transfected with reporter plasmid only and mock-infected, served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are expressed as mean ± standard deviation of normalized luciferase activity. *P <0.05 relative to RSV or hMPV infected cells. (B) Nuclear extracts prepared from A549 cells infected with RSV (left panel) or hMPV (right panel) for various periods of time post infection (p.i.) were subjected to western blot with anti Nrf2 antibody. Membranes were stripped and reprobed for lamin B as an internal control for protein integrity and loading.

Figure 2. Schematic representation of the proposed mechanisms of oxidative cell damage during RSV infection

RSV infection of airway epithelial cells leads to increased superoxide formation and increased H₂O₂ production, due to up regulation of SOD 2 expression and activity. RSV-induced inhibition of Nrf2 activation, due to proteasome-dependent degradation, causes a progressive decrease in the expression of a variety of AOEs involved in H₂O₂ detoxification leading to accumulation of highly reactive radicals, such as hydroxyl radical, and subsequent cellular damage (* autoxidation in presence of transition metals).

Figure 3. Effect of EUK treatment on RSV-induced ROS formation and oxidative stress

(A) A549 cells were treated with different micromolar concentrations of EUK-8 and EUK-189, infected with RSV for 24 h, and harvested to measure DCF-DA fluorescence. Ctrl indicates uninfected cells. Mean Fluorescence Intensity is reported as percent increase over control. (B) Cell supernatants were harvested at 24 h p.i. to measure F2-isoprostanes. Results are expressed as mean ± standard error. Results are representative of two independent experiments run in triplicate. *p<0.05, **p<0.01 compared to untreated RSV-infected cells. Reprinted with permission of the American Physiological Society. Copyright © 2014 The American Physiological Society [78].