p53 as a retrovirus-induced oxidative stress modulator

Abstract

Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus, ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared with uninfected astrocytes. Previously, we have demonstrated that ts1 infection caused two separate events of ROS upregulation. The first upregulation occurs during early viral establishment in host cells and the second during the virus-mediated apoptotic process. In this study, we show that virus-mediated ROS upregulation activates the protein kinase, ataxia telangiectasia mutated, which in turn phosphorylates serine 15 on p53. This activation of p53 however, is unlikely associated with ts1-induced cell death. Rather p53 appears to be involved in suppressing intracellular ROS levels in astrocytes under oxidative stress. The activated p53 appears to delay retroviral gene expression by suppressing NADPH oxidase, a superoxide-producing enzyme. These results suggest that p53 plays a role as a retrovirus-mediated oxidative stress modulator.

Fig. 1.

ATM activates p53 in response to intracellular ROS upregulation and ts1 infection. To access activation of ATM and p53, C1 cell lysates were harvested and subjected to immunoblotting using anti-ATM and anti-p53 antibodies after the following treatment: (a) 100 µM H₂O₂ treatment, (b) 100 µM H₂O₂ treatment with or without 5 mM Ku55933, (c) ts1 infection, (d) ts1 infection with or without 5 mM Ku55933 [C1 astrocytes were infected with ts1 (m.o.i. 5) as described previously (Kim & Wong, 2013)], (e) 0.5 µg tunicamycin ml⁻¹. To verify unfolded protein response, immunoblotting was performed using anti-BiP antibody.

Fig. 2.

p53 is involved in antioxidant response rather than ts1-induced apoptosis. (a) Survival curves of p53^+/+ and p53^−/− mouse after ts1 infection. The disease progression was divided into four stages as described in the text and mice were sacrificed at stage IV (days p.i., days post-infection). (b) PAC viability counts after ts1 infection. PAC were isolated from 1 to 2-day-old newborn mouse pups at an m.o.i. of 20 by a method described previously (Jiang et al., 2006). Pup genotypes (p53^+/+ or p53^−/−) were identified using standard genotyping PCR on tissue samples (ns, not significant). Intracellular ROS levels were measured using dichlorodihydrofluorescein (DCF) fluorescence (c) in the absence of H₂O₂ (d) or in the presence of 100 µM H₂O₂. (d) The ROS levels in H₂O₂ treated cells were compared with those in the same genotype cells without H₂O₂ treatment (100 %) and presented as relative amount (%). Statistical significance was determined by Student’s t-test (*P = 0.0366, **P = 0.001, ****P<0.0001). (e) Intracellular $O_2^{-}$ levels were detected by loading with 1 mg dihydroethidium (DHE) ml⁻¹ in PBS for one hour as described previously (Behrens et al., 2007). Intracellular $O_2^{-}$ levels in ts1-infected cells were compared with that in the same genotype cells without ts1 infection and presented as relative amount. **P = 0.0063. All in vitro PAC experiments were performed at least three times and all experiments were duplicated.

Fig. 3.

p53 plays a role in suppressing NADPH oxidase expression resulting in delayed viral protein expression. (a–c) PAC were isolated from 1 to 2-day-old newborn mouse pups at an m.o.i. of 20 by a method described previously (Jiang et al., 2006). p53^+/+ and p53^−/− PACs lysates harvested after ts1 infection and subjected to immunoblotting using indicated antibodies described in the text. gpr80^env, gpr70^env and p30^capsid are the precursor and mature form of the envelope, and capsid protein. gp91^phox is the catalytic subunit of NADPH oxidase complex. Each band represents 1, 2, 3, 4, 5 and 6 days p.i.