The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism

Abstract

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

FIG. 1.

The presence of P58^IPK increases viral mRNA translation during influenza virus infection. P58^IPK KO and WT MEFs were mock infected or infected with the WSN strain of influenza virus at an MOI of 2 PFU/cell. (A) Cells were labeled with [³⁵S]methionine for 30 min at the indicated times p.i. Cells were lysed, and labeled proteins were analyzed by SDS-10% PAGE and autoradiography. The positions of the major viral proteins NP, M1, and NS1 are indicated. (B) Densitometry analysis of three independent experiments. The density of the M1-plus-NS1 band was normalized to the density of the entire lane, with each bar representing the mean ± standard deviation. (C) Total RNA was isolated from the cells at 8 h p.i. and reverse transcribed to generate cDNA. Quantitative RT-PCR was used to determine the amount of viral M1 and NP mRNA in each sample.

FIG. 2.

The lack of P58^IPK causes increased eIF2α phosphorylation during influenza virus infection. (A) P58^IPK KO and WT MEFs were mock infected or infected with the WSN strain of influenza virus at an MOI of 2 PFU/cell. Cells were lysed at the indicated times p.i., and equivalent concentrations of protein were subjected to SDS-12.5% PAGE. The levels of phosphorylated eIF2α, total eIF2α, and actin were determined by immunoblot analysis. (B) Densitometry analysis of two independent experiments. The densities of the eIF2α or eIF2α-P bands were normalized to their corresponding actin bands. Total eIF2α phosphorylation was determined by dividing the normalized eIF2α-P value by the eIF2α value, with each bar representing the mean ± standard deviation.

FIG. 3.

The lack of P58^IPK causes increased PKR phosphorylation during influenza virus infection. (A) P58^IPK KO and WT MEFs were mock infected or infected with the WSN strain of influenza virus at a MOI of 1, 10, or 100 PFU/cell. Cells were lysed at 8 h p.i., and equivalent concentrations of protein were subjected to SDS-10% PAGE. The levels of phosphorylated PKR, total PKR, and actin were determined by immunoblot analysis. (B) Densitometry analysis. The density of the P-PKR band was normalized to the density of the PKR band, which is represented graphically.

FIG. 4.

VSV replication is more efficient in the presence of P58^IPK. P58^IPK KO and WT MEFs were infected with VSV at an MOI of 10, 1, or 0.1 PFU/cell. At 24 h p.i., progeny virion production was determined by standard plaque assay on BHK-21 cells. Each result represents the mean activity for two independent experiments ± standard deviation.

FIG. 5.

The absence of P58^IPK results in increased reovirus replication. P58^IPK KO (dotted line) and WT (solid line) MEFs were infected with reovirus strain Dearing, c8, or c87 at a multiplicity of 2 PFU/cell. Infectious virus present at 0, 1, 3, and 5 days p.i. was measured by plaque assay on L929 cells. Each result represents the mean activity for three independent experiments ± standard deviation. P values from a two-tailed t test assuming nonequal variance are indicated (*, P ≤ 0.05; **, P ≤ 0.005).

FIG. 6.

The lack of PKR increases viral mRNA translation during influenza virus infection. PKR KO and WT MEFs were mock infected or infected with the WSN strain of influenza virus at an MOI of 1 PFU/cell. (A) Cells were labeled with [³⁵S]methionine for 30 min at the indicated times p.i. Cells were lysed, and labeled proteins were analyzed by SDS-10% PAGE and autoradiography. The positions of the major viral proteins NP, M1, and NS1 are indicated. (B) Densitometry analysis for three independent experiments. The density of the NP band was normalized to the density of the entire lane, with each bar representing the mean ± standard deviation.

FIG. 7.

The presence of PKR causes increased eIF2α phosphorylation during influenza virus infection. (A) PKR KO and WT MEFs were mock infected or infected with the WSN strain of influenza virus at an MOI of 1 PFU/cell. Cells were lysed at the indicated times p.i., and equivalent concentrations of protein were subjected to SDS-12.5% PAGE. The levels of phosphorylated eIF2α, total eIF2α, and actin were determined by immunoblot analysis. (B) Densitometry analysis. The densities of the eIF2α or eIF2α-P bands were normalized to their corresponding actin bands. Total eIF2α phosphorylation was determined by dividing the normalized eIF2α-P value by the eIF2α value, which is represented graphically.

FIG. 8.

The lack of phosphorylatable eIF2α causes decreased levels of influenza virus protein synthesis and viral mRNA. eIF2αS51A and WT MEFs were mock infected or infected with the WSN strain of influenza virus at an MOI of 1 PFU/cell. (A) Cells were labeled with [³⁵S]methionine for 30 min at the indicated times p.i. Cells were lysed and analyzed by SDS-10% PAGE and autoradiography. The positions of major viral proteins NP, M1, and NS1 are indicated. (B) Densitometry analysis for three independent experiments. The density of the NP band was normalized to the density of the entire lane, with each bar representing the mean ± standard deviation. (C) Total RNA was isolated from the cells at 9 h p.i. and reverse transcribed to generate cDNA. Quantitative RT-PCR was used to determine the amount of viral M1 and NP mRNA in each sample. (D) Cell lysates from the experiment in panel A were analyzed by SDS-12.5% PAGE. The levels of phosphorylated eIF2α and total eIF2α were determined by immunoblot analysis.

FIG. 9.

Model of the role of P58^IPK during influenza virus infection. In the presence of influenza virus infection, PKR and P58^IPK are activated. In the presence of P58^IPK, PKR inhibition is increased, resulting in decreased levels of eIF2α phosphorylation, antiviral genes, and apoptosis, resulting in increased influenza virus mRNA translation. When PERK is absent or eIF2α cannot be phosphorylated, ER homeostasis is compromised, resulting in poor viral mRNA translation in relation to decreased eIF2α phosphorylation levels. During the viral life cycle, changes in viral mRNA translation will amplify changes in viral replication. See the text for further details.