Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71

Abstract

Viruses have been known to subvert the anti-apoptotic pathways of the host cell in order to delay apoptosis. However, the mechanisms utilized by enterovirus 71 (EV71) to mediate anti-apoptotic activity remained undetermined. We observed that EV71 infection induced an early activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK/ERK signaling pathways. The activity of GSK3beta, a downstream target of these pathways, was negatively regulated by the activation of both MAPK/ERK and PI3K/Akt. The phosphorylation of GSK3 could be inhibited by treatment with the specific inhibitors of MAPK/ERK and PI3K/Akt. Other Akt downstream targets, BAD, caspase-9 and the Forkhead transcription factor (FKHR), were not phosphorylated during the course of infection by EV71. We further demonstrated that infection by UV-irradiated, inactivated virus triggered early Akt activation but was insufficient to trigger late Akt activation. These data suggest that with the phosphorylation of MAPK/ERK and PI3K/Akt the subsequent inactivation of GSK3beta is utilized by EV71 as a potential mechanism to delay host cell apoptosis.

Fig. 1

Time course for EV-71 stimulation and Akt phosphorylation. Growth-arrested Vero (A–C) and MRC-5 (D and E) cells were incubated with EV-71 at an MOI of 5 for 1 h and then the cells were washed with PBS twice and replenished with serum-free medium. For immunoblot analysis, cell lysates were collected at the indicated times following EV-71 infection and equal amounts of protein were subjected to SDS-12% PAGE. Proteins were transferred onto nitrocellulose membranes and subjected to Western blotting. Akt activity was analyzed based on phosphorylated Akt (P-Akt). Unphosphorylated Akt was used as the loading control. The control samples contained the same serum content as the virus group. In C, Vero cells were subjected to immunofluorescent staining at 0.5 h pi. Akt redistribution was indicated by arrows. The immunoblot results were quantitated by densitometric analysis and normalized to control 0.5 h levels arbitrarily set to 1 (B and E). Values are means ± S.E.M. from three independent experiments for B and E. *P < 0.05 compared with respective control.

Fig. 2

EV71 strongly promotes expression of phospho-Akt and phosphor-GSK-3 but not phospho-caspase 9 and phospho-FKHR. (A) Vero cells were serum-starved overnight, induced with EV71 and harvested at the indicated time points. The phosphorylation of caspase 9 and FKHR was detected by Western blot using respective antibodies. (B and C) GSK phosphorylation was monitored with polyclonal antibodies against phosphorylated GSK (Ser9). Vero cells were pre-treated with or without wortmannin (0.1 μM, Lane 3) for 3 h and PD98059 (PD, 50 μM, Lane 4) for 1 h, followed by infection with EV71 (MOI5) to induce phosphorylation of Akt and GSK3β. Unphosphorylated Akt was used as the control. The results were also quantitated and normalized as in Fig. 1. Values are means ± S.E.M. from three independent experiments. *P < 0.05 compared with control; ^¥P < 0.05 compared with virus infection alone.

Fig. 3

Time course for EV-71 stimulation of ERK1/2 phosphorylation. Growth-arrested Vero (A and B) and MRC-5 (C and D) cells were incubated with EV71 at an MOI of 5 for 1 h. The cells were then washed with PBS twice and replenished with serum-free medium. Cell lysates were collected at the indicated times following EV71 infection and were processed as in Fig. 1. ERK1/2 activity was determined based on phosphorylated ERK1/2 (P-ERK1/2). In the lower panels, the results were quantitated and normalized as in Fig. 1. Values are means ± S.E.M. from three independent experiments. *P < 0.05 compared with control.

Fig. 4

Early- but not late-phase Akt phosphorylation caused by UV-inactivated EV71. The virus was UV-irradiated for 1 h and the remaining titer of the inactivated virus was determined by the plaque assay. The UV-irradiated virus lost infection activity completely after treatment (data not shown). Vero cells were infected with either wild-type virus or UV-inactivated virus. Following 0, 10, 20, 30, and 60 min (A and B) or 0.5, 3 and 6 h (C and D) after infection, cell lysates were harvested and Western blotting was performed to determine Akt activation. Unphosphorylated Akt was used as the control. Values are means ± S.E.M. from four independent experiments. * and ^¥, P <0.05 compared with respective control.