SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

Abstract

Viral infections sustain their replication cycle promoting a pro-oxidant environment in the host cell. In this context, specific alterations of the levels and homeostatic function of the tripeptide glutathione have been reported to play a causal role in the pro-oxidant and cytopathic effects (CPE) of the virus. In this study, these aspects were investigated for the first time in SARS-CoV2-infected Vero E6 cells, a reliable and well-characterized in vitro model of this infection. SARS-CoV2 markedly decreased the levels of cellular thiols, essentially lowering the reduced form of glutathione (GSH). Such an important defect occurred early in the CPE process (in the first 24 hpi). Thiol analysis in N-acetyl-Cys (NAC)-treated cells and membrane transporter expression data demonstrated that both a lowered uptake of the GSH biosynthesis precursor Cys and an increased efflux of cellular thiols, could play a role in this context. Increased levels of oxidized glutathione (GSSG) and protein glutathionylation were also observed along with upregulation of the ER stress marker PERK. The antiviral drugs Remdesivir (Rem) and Nelfinavir (Nel) influenced these changes at different levels, essentially confirming the importance or blocking viral replication to prevent GSH depletion in the host cell. Accordingly, Nel, the most potent antiviral in our in vitro study, produced a timely activation of Nrf2 transcription factor and a GSH enhancing response that synergized with NAC to restore GSH levels in the infected cells. Despite poor in vitro antiviral potency and GSH enhancing function, Rem treatment was found to prevent the SARS-CoV2-induced glutathionylation of cellular proteins. In conclusion, SARS-CoV2 infection impairs the metabolism of cellular glutathione. NAC and the antiviral Nel can prevent such defect in vitro.

Keywords: COVID-19; Glutathione; Nrf2; Protein glutathionylation; SARS-CoV-2; Thiols.

Graphical abstract

Fig. 1

Levels of thiols and protein glutathionylation in SARS-CoV2 infected VERO E6 cells treated with Remdesivir (Rem) and Nelfinavir (Nel) antiviral compounds. The cells were grown in complete medium and infected with SARS-CoV2 (MOI:0.0035) as described in the section Methods. Treatments with Rem and Nel utilized at the final concentrations of 1 μM and 5 μM (in DMSO, 0.001% vol/vol) lasted 24 hpi. Total intracellular thiols (A) were measured by Ellman's test using 100 μg of protein extract of VERO E6 cells. Intracellular (B) and extracellular (C) levels of GSH and other thiols measured by HPLC 24 hpi. in cells treated with Rem or Nel. S-glutathionylation (PSSG) of cellular proteins assessed 24 hpi by immunoblot (D) or by semi-quantitative immunofluorescence (PSSG-FITC, green; DAPI for nuclei, blue) (E). Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals: *p < 0.05, **p < 0.001.

Fig. 2

Effect of NAC on cellular thiols of SARS-CoV2 infected VERO-E6 cells. VERO E6 cells were treated with 5 mM NAC utilizing two different protocols: in the pre-NAC protocol the cells were treated in complete medium (CM) with NAC before infection and then NAC-containing medium was replaced with a medium deprived of sulfur-containing amino acids (non-complete medium or NCM) for infection; in the co-NAC protocol the cells were directly exposed to NAC in NCM during the infection (A). Thiols were assessed 24 hpi. intracellular GSH (B); intracellular Cys (C); Intracellular GSSG (D); extracellular GSH (E); extracellular Cys (F) and extracellular CysGly (G). NCM vs co-NAC or pre-NAC: a (control test, black bars) and b (SARS-CoV2 infected cells, white bars): p < 0.05; control cells (black bars) vs SARS-CoV2 infected cells (white bars) in the same NAC treatment conditions: *p < 0.05; **p < 0.001.

Fig. 3

Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression (A, left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi (A, right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins (B), and GCLC protein (C) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of Fig. 1. Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Expression of the stress proteins NQO1, GSTP and PERK in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Qualitative and semi-quantitative fluorescence analysis of NQO1-Cy5 (red) (A) and GSTP-Texas Red (red) (B) were measured 24 hpi. Nuclei were labelled with DAPI (blue) and cytosol with Phalloidin -Alexa Fluor488 (green). Immunoblot of PERK protein expression was assessed by immunoblot 6 hpi and 24 hpi (C). Infection conditions and treatments with antivirals were the same of Fig. 1. Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)