Reovirus infection activates JNK and the JNK-dependent transcription factor c-Jun

Abstract

Viral infection often perturbs host cell signaling pathways including those involving mitogen-activated protein kinases (MAPKs). We now show that reovirus infection results in the selective activation of c-Jun N-terminal kinase (JNK). Reovirus-induced JNK activation is associated with an increase in the phosphorylation of the JNK-dependent transcription factor c-Jun. Reovirus serotype 3 prototype strains Abney (T3A) and Dearing (T3D) induce significantly more JNK activation and c-Jun phosphorylation than does the serotype 1 prototypic strain Lang (T1L). T3D and T3A also induce more apoptosis in infected cells than T1L, and there was a significant correlation between the ability of these viruses to phosphorylate c-Jun and induce apoptosis. However, reovirus-induced apoptosis, but not reovirus-induced c-Jun phosphorylation, is inhibited by blocking TRAIL/receptor binding, suggesting that apoptosis and c-Jun phosphorylation involve parallel rather than identical pathways. Strain-specific differences in JNK activation are determined by the reovirus S1 and M2 gene segments, which encode viral outer capsid proteins (sigma1 and mu1c) involved in receptor binding and host cell membrane penetration. These same gene segments also determine differences in the capacity of reovirus strains to induce apoptosis, and again a significant correlation between the capacity of T1L x T3D reassortant reoviruses to both activate JNK and phosphorylate c-Jun and to induce apoptosis was shown. The extracellular signal-related kinase (ERK) is also activated in a strain-specific manner following reovirus infection. Unlike JNK activation, ERK activation could not be mapped to specific reovirus gene segments, suggesting that ERK activation and JNK activation are triggered by different events during virus-host cell interaction.

FIG. 1

Reovirus activates JNK infected cells. L929 cells were infected with two different serotypes of reovirus, T1L and T3D (MOI, 100) or were mock infected. At various times p.i., lysates were prepared and JNK activity was determined by in vitro kinase assays. The graph shows the mean JNK activity, as measured by c-Jun phosphorylation (arbitrary imager units), of three independent experiments. Error bars represent standard errors of the mean.

FIG. 2

c-Jun is activated following infection with reovirus. Cells were infected with different strains of reovirus (MOI, 100) and were harvested at various times p.i. (A and C) Extracts were standardized for protein concentration, using an anti-actin antibody, and equal amounts of protein were separated by SDS-PAGE and probed with antibodies directed against phosphorylated (A) or total (C) c-Jun. Bands corresponding to phosphorylated and unphosphorylated c-Jun are shown. The gels are representative of at least two independent experiments. (B) Graphical representation of the Western blot shown in panel A, showing the fold increase in the levels of phophorylated c-Jun over time.

FIG. 3

There is a correlation between the capacities of different prototype strains of reovirus to phosphorylate c-Jun and induce apoptosis and between the capacities of T1L × T3D reassortant viruses to induce JNK activation or c-Jun phosphorylation and apoptosis. The abilities of different strains of reovirus (T3A, T3D, and T1L) and mock (M) infection to induce increased levels of phosphorylated c-Jun, at 12 h (A) and 18 h (B) p.i. (values taken from Fig. 2) and apoptosis are shown. Experiments to determine c-Jun phosphorylation and apoptosis were set up in parallel. (C) The capacity of reovirus reassortants (MOI, 100) to induce phosphorylated c-Jun and apoptosis was plotted. Each point represents a single reassortant virus. Lysates were harvested at 18 h p.i., standardized for protein concentration, and analyzed by Western blotting using an antibody directed against phospho c-Jun. Apoptosis values were obtained in a parallel experiment and represent the mean value from three separate wells (24-well tissue culture plate) of the same experiment. (D) The capacities of reovirus reassortants (MOI, 100) to induce JNK activity and apoptosis were plotted. Each point represents a single reassortant virus. JNK activity values were taken from Table 1. Apoptosis values were obtained in a parallel experiment and represent the mean value from three separate wells (24-well tissue culture plate) of the same experiment.

FIG. 4

Apoptosis but not c-Jun phosphorylation is inhibited in T3D-infected cells in the presence of the soluble TRAIL receptors Fc:DR5 and Fc:DR4. In parallel experiments, cells were infected with T3D (MOI, 50) and were assayed for c-Jun activation at various times p.i. and for apoptosis after 48 h. (A) Representative autoradiograph showing levels of phosphorylated c-Jun following infection with reovirus (T3D), in the presence or absence of Fc:DR5 and Fc:DR4 (final concentrations, 100 ng/ml each). Equal amounts of protein, as determined by actin concentration (data not shown), were loaded. (B) Graphical analysis of the results shown in panel A showing the fold increase in c-Jun phosphorylation compared to that in mock-infected, untreated cells, at 12 h, 20 h, and 30 h p.i. (C) Graph showing the percentage of cells with apoptotic nuclear morphology in reovirus (T3D)- or mock-infected cells in the presence or absence of soluble TRAIL receptors (final concentration, 100 ng/ml each). Error bars represent standard errors of the mean from three separate wells (24-well tissue culture plate) of the same experiment.

FIG. 5

Reovirus activates ERK but not p38 in infected cells. (A and B) The activities of ERK (A) and p38 (B) were investigated in reovirus-infected L929 cells (MOI, 100) at 24 and 48 h p.i. by in vitro kinase assays. The graphs show fold activation compared to that in mock-infected cells. Error bars represent standard errors of the mean from three independent experiments. (C) ERK is activated at early times after reovirus infection. L929 cells were infected with reovirus (MOI, 100) and harvested at various times p.i. Proteins were separated by SDS-PAGE and subjected to Western blot analysis using antibodies directed against phospho-ERK. Actin was used to standardize for protein loading (data not shown).

FIG. 6

Inhibition of ERK activation does not affect reovirus-induced apoptosis. (A) Reovirus-induced apoptosis (MOI, 100) was measured in the presence of a chemical inhibitor of ERK activation (PD 98059). L929 cells were pretreated with inhibitor (10 μM) for 2 h prior to infection with reovirus and were maintained in medium in the presence of inhibitor for 48 h following reovirus infection. They were then harvested and assayed for apoptosis. The graph shows percent apoptosis. Error bars represent standard errors of the mean. (B) Activation-phosphorylation of ERK following reovirus (T3D) infection (MOI, 100) in the presence of PD 98059 (10 μM) as determined by Western blot analysis using antibodies directed against phospho-ERK.