Reovirus induces and benefits from an integrated cellular stress response

Abstract

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58(IPK), the cellular inhibitor of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2alpha phosphorylation and unphosphorylated eIF2alpha is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2alpha phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2alpha kinases and phosphorylatable eIF2alpha. When eIF2alpha is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2alpha phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2alpha phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

FIG. 1.

Reovirus strains vary in their effects on host cells. (A) L929 cells were mock infected or infected in triplicate with the indicated reovirus strains at a multiplicity of 80 PFU/cell. At 19.5 h p.i., cells were pulse labeled with [³⁵S]methionine-cysteine for 30 min. Extracts were prepared, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Reovirus proteins are labeled with brackets to the right of the gel image. (B) L929 cells were mock infected or infected in triplicate with the indicated strains of reovirus at an MOI of 80 PFU/cell. At 1, 6, 12, and 20 h p.i., total RNAs were extracted, and the levels of IFN-β mRNA were measured by real-time reverse transcription-PCR. The relative increase in IFN-β mRNA compared to that in mock-infected cells at each time p.i. was calculated. Each bar represents the average (± SD) from three samples. (C) L929 cells were mock infected or infected in triplicate with the indicated reovirus strains at a multiplicity of 80 PFU/cell. At 19.5 h p.i., cells were harvested, stained with propidium iodide and annexin V-biotin, sorted by flow cytometry using a FACSCalibur machine, and analyzed with FLOWJO software. Each bar represents the average from three samples. PI-AV− indicates viable cells, PI−AV+ indicates early apoptotic cells, PI+AV+ indicates late apoptotic/necrotic cells, and PI+AV− indicates necrotic cells.

FIG. 2.

Reovirus-induced changes in levels of L929 transcripts. (A) Number of genes on the Affymetrix U74Av2 chip that increased (black portion of each bar) or decreased (gray portion of each bar) 2-, 2.5-, 3-, or 5-fold, with all P values being ≤0.05, after infection with at least one of the three strains of reovirus examined. (B and C) Venn diagrams of cellular genes whose expression increased (B) and decreased (C) ≥3-fold, illustrating which infection conditions (light gray, one reovirus strain; gray, two viruses; and dark gray, all three reoviruses) altered the level of cellular transcripts. Ten genes of the 344 total were included in more than one category because they were represented by more than one probe and the intensity values for the different probes were not identical.

FIG. 3.

P58^IPK expression changes in reovirus-infected cells. L929 cells were treated with tunicamycin (Tu), mock infected, or infected with the indicated strains of reovirus at an MOI of 80 PFU/cell. (A) At 19.5 h p.i., total RNAs were isolated, and the amounts of P58^IPK mRNA were analyzed by Northern blotting (top panel). The blot was then stripped and reprobed with a GAPDH mRNA-specific probe (bottom panel). (B) At 19.5 h p.i., cell extracts were harvested, and P58^IPK protein in 25 μg of cell extract was detected by immunoblot analysis.

FIG. 4.

Effect of PERK on reovirus replication and role of PERK in reovirus-induced host shutoff. Primary (A) and immortalized (B) wt and PERK KO MEFs were infected with the indicated strains of reovirus at a multiplicity of 2 PFU/cell. Virus yields at 0, 1, 3, and 5 days p.i. were measured by a plaque assay. Each time point represents the mean (± SD) derived from three samples. Primary wt (C) and PERK KO (D) MEFs were mock infected or infected in triplicate with the indicated reovirus strains at a multiplicity of 80 PFU/cell. Cells were metabolically labeled at 19.5 h p.i. with [³⁵S]methionine-cysteine for 2 h, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Reovirus proteins are labeled with brackets to the right of the gel image. Percentages underneath the gel images represent the percentage of cellular translation relative to the level in uninfected cells quantified from the area indicated by the left bracket ± SD.

FIG. 5.

Reovirus replication in MEFs that have constitutively active eIF2α. wt MEFs and MEFs expressing S51A mutant eIF2α were infected with the indicated strains of reovirus at a multiplicity of 2 PFU/cell. Amounts of infectious virus present at 0, 1, 3, and 5 days p.i. were determined as described in the legend to Fig. 4.

FIG. 6.

ATF4 expression in reovirus-infected cells and its impact on reovirus replication. (A and B) L929 cells were mock infected or infected with the indicated strains of reovirus at an MOI of 80 PFU/cell, and cell extracts were harvested at 19.5 h p.i. Samples were adjusted to contain 20 μg of protein for eIF2α analysis and 25 μg of protein for ATF4 analysis. The levels of phosphorylated eIF2α (A, top), total eIF2α (A, bottom), and ATF4 (B) were examined by immunoblot analysis. The band intensities for phosphorylated and total eIF2α were determined using NIH Image. The ratio of phosphorylated eIF2α to total eIF2α was set at 1 for mock-infected cells, and relative ratios for the infected samples are displayed beneath panel A. (C) Primary wt and ATF4 KO MEFs were infected with the indicated strains of reovirus at an MOI of 2 PFU/cell. The amounts of infectious virus present at 0, 1, 3, and 5 days p.i. were determined as described in the legend to Fig. 4.

FIG. 7.

Stress granule formation in reovirus-infected cells. DU145 cells were mock infected (A), treated with sodium arsenite (B), or infected with Dearing (C), c8 (D), or c87 (E) at a multiplicity of 80 PFU/cell. At 19.5 h p.i., cells were fixed, and the intracellular localization of TIAR and nuclei was determined by indirect immunofluorescence.