Loss of GSH, Oxidative Stress, and Decrease of Intracellular pH as Sequential Steps in Viral Infection

Maria Rosa Ciriolo,Anna Teresa Palamara,Sandra Incerpi,Emanuela Lafavia,Maria Cristina Buè,Paolo De Vito,Enrico Garaci,Giuseppe Rotilio

doi:10.1074/jbc.272.5.2700

Maria Rosa Ciriolo, Anna Teresa Palamara + Show 6 more

Open Access

https://doi.org/10.1074/jbc.272.5.2700

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Abstract

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Highlights

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products
In the present report we investigated the replication of Sendai virus in Madin-Darby canine kidney (MDCK) cells
The results reported above demonstrate that GSH depletion is a direct consequence of viral infection

Summary

EXPERIMENTAL PROCEDURES

Cells and Reagents—MDCK cells were purchased from the American Type Culture Collection and grown in RPMI 1640 supplemented with heat-inactivated 5% fetal calf serum, 0.3 mg/ml glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Titration of purified virus was performed by measuring the number of hemagglutinin units present in the medium of infected monolayers of MDCK cells at different times postinfection. At the end of the treatments, culture medium was collected and utilized for virus titration, and cells were washed, scraped, and pelleted by centrifugation at 700 ϫ g for 10 min and treated for GSH determination. In order to rule out the contribution of HCO3Ϫ-dependent transport mechanisms [30], all experiments were carried out in bicarbonate-free buffer having the following composition: 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 20 mM HEPES, pH 7.3 This buffer was used for the incubation with the fluorescent probe and for the determination of intracellular pH, unless otherwise stated; the cells incubated in this buffer were considered virtually depleted of bicarbonate. Incubation with the fluorescent dye was carried out as previously reported [31]; cells were washed twice with Naϩ buffer, and under these

GSH Loss and Viral Replication

RESULTS

TABLE II Glutathione status of MDCK cells during Sendai virus replication

DISCUSSION

Viral proteins