The Leader Protein of Theiler's Virus Prevents the Activation of PKR

Abstract

Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler's murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA.IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler's virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

Keywords: PKR; Theiler's murine encephalomyelitis virus; cardiovirus; double-stranded RNA virus; leader protein; picornavirus.

FIG 1

TMEV-induced stress granule assembly correlates with eIF2α phosphorylation and PKR activation. (A) Confocal microscopy images showing the coimmunostaining of eIF3 (green) and of the viral capsid protein VP1 (red) in HeLa cells infected for 10 h with the wild-type virus (L^WT) or L-mutant viruses (L^Zn and L^M60V). Note that some VP1-negative cells also display SG in the wells infected with the L-mutant viruses. This is probably due to the fact that VP1 had not reached a detectable level in some infected cells at the time the cells were fixed. (B) Western blot analysis of eIF2α and PKR phosphorylation in HeLa cells infected as described for panel A. 3D is the viral polymerase; β-actin detection was used as an additional loading control.

FIG 2

Pharmacological inhibition of PKR prevents TMEV-induced eIF2α phosphorylation and stress granule assembly. HeLa cells were treated with either DMSO or C16 and infected for 10 h with the wild-type virus (L^WT) or L-mutant viruses (L^Zn and L^M60V). As a control, DMSO- and C16-treated cells were treated with sodium arsenite (Ars) to induce PKR-independent phosphorylation of eIF2α. (A) Western blot analysis of eIF2α and PKR phosphorylation. (B) Confocal microscopy images showing the coimmunostaining of eIF3 (green) and the viral capsid (red).

FIG 3

PKR is responsible for TMEV-induced eIF2α phosphorylation and stress granule assembly. (A) Western blot analysis of PKR expression in HeLa cells transduced with an empty pLKO-1 lentiviral vector (control) or with lentiviral vectors (FB52, FB53, and FB54) expressing shRNA1, shRNA2, or shRNA3, directed against PKR. Transduced cells were selected for 1 week with puromycin before being assessed for PKR expression. (B) Control HeLa cells and HeLa cells transduced with FB54 (shRNA3) were infected for 10 h with wild-type (L^WT) or L-mutant (L^Zn and L^M60V) viruses. Mock-infected and L^M60V-infected cell samples were treated for 30 min with sodium arsenite prior to cell lysis. Lysates were harvested at 10 hpi, and PKR expression and eIF2α phosphorylation were analyzed by Western blotting. (C) Confocal microscopy images showing the coimmunostaining of eIF3 (green) and the viral capsid (red) in control and PKR knockdown HeLa cells infected or treated as described for panel B.

FIG 4

L acts at two levels in the stress granule formation pathway. (A to D) L^WT prevents NaAsO₂-mediated SG assembly. HeLa cells were left noninfected (NI) of were infected for 10 h with KJ6 (L^WT). Where indicated, cells were treated for 30 min with NaAsO₂ before being fixed and processed for eIF3 and VP1 immunostaining (A and D) or before protein harvest and Western blot analysis of eIF2α phosphorylation (B and C). (A) Representative confocal microscopy images showing the absence of stress granules in L^WT-infected NaAsO₂-treated cells (upper right). (B) Representative Western blot monitoring of eIF2α (Ser51) phosphorylation and total eIF2α in uninfected and infected cells treated with increasing concentrations of NaAsO₂. (C) Quantification (mean and standard deviation [SD] values) of the data shown in panel B (n = 3). (D) Percentage (mean and SD values) of SG-positive cells, among infected cells, as counted from microscopy images taken from uninfected or infected NaAsO₂-treated cells (n = 3). (E and F) TMEV and mengovirus L^WT proteins can inhibit PKR activation. HeLa cells were infected with L^WT or the indicated L mutant viruses (12 h, 2 PFU per cell for TMEV; 6 h, 5 PFU per cell for mengovirus). Phospho-PKR (Thr446) and total PKR were quantified by Western blotting (mean and SD values; n = 5 for TMEV, n = 4 for mengovirus). *, P < 0.05; **, P < 0.01; ***, P < 0.001; all by analysis of variance comparisons.

FIG 5

PKR is a strong antagonist of TMEV replication. Control- or PKR^Knockdown-HeLa cells were infected with wild-type virus (L^WT) or L-mutant viruses (L^Zn and L^M60V), and supernatants were harvested at 16 h postinfection. Viral titers were measured by plaque assay. Histograms show the mean and SD values from three independent infection experiments.

FIG 6

L prevents the interaction between viral dsRNA and PKR. (A) HeLa cells were infected with 5 PFU per cell of TM994, TM1016, and TM1017, recombinant TMEV expressing 3×FLAG-L^WT, 3×FLAG-L^Zn, and 3×FLAG-L^M60V, respectively. At 10 hpi, cells were lysed and 3×FLAG-L proteins were immunoprecipitated using an anti-FLAG antibody. The presence of L (FLAG detection), PKR, phospho-PKR (Thr446), and G3BP1 was assessed by Western blotting in cell lysates (ly), in postimmunoprecipitation lysate fractions (ly_p), and in immunoprecipitates (IP). (B) Confocal microscopy images showing the coimmunostaining of dsRNA and PKR in HeLa cells infected for 10 h with wild-type (L^WT) or L-mutant (L^Zn and L^M60V) viruses. Note the marked redistribution of PKR to punctate structures in cells infected with the L-mutant viruses. (C) Confocal microscopy images showing the coimmunostaining of PKR, eIF3, and G3BP (left) and of PKR, eIF3, and dsRNA (right) in HeLa cells infected, as described for panel B, with the L^M60V mutant virus. (D) Cartoon summarizing the observations. (E) HeLa cells were infected for 10 h with the wild-type or L-mutant viruses or transfected with poly(I·C) for 4 h before lysis and dsRNA immunoprecipitation. The presence of PKR, phospho-PKR, and L was monitored by Western blotting in equivalent amounts of whole-cell lysates and IP products. Graphs show the mean and SD values of PKR amounts detected in lysates and dsRNA-immunoprecipitated fractions from 3 experiments. *, P < 0.05 by analysis of variance pairwise comparisons between lysates and IP fractions.

FIG 7

L affects the responsiveness of PKR to dsRNA. (A and B) HeLa cells were infected with the wild-type (L^WT) and L^Zn mutant viruses. At 6 hpi, cells were transfected with 500 ng of poly(I·C) or control treated with the transfection reagent only. At 10 hpi, whole-cell lysates were harvested and analyzed by Western blotting to monitor PKR phosphorylation on threonine 446 and 451. (B) Quantification of the phospho-Thr446/total PKR ratio from 3 experiments (mean and SD values). *, P < 0.05; **, P < 0.01; ***, P < 0.001; all by analysis of variance comparisons. (C and D) HeLa cells were infected for 10 h with the wild-type virus (L^WT) or with a mutant virus that expresses GFP instead of L (ΔL-GFP) or were coinfected with both viruses. PKR phosphorylation was assessed by Western blotting (C), and its distribution was monitored by classical immunofluorescence microscopy (D). White arrows point to some infected cells presenting a typical punctate redistribution of PKR.