Induction of protein kinase PKR-dependent activation of interferon regulatory factor 3 by vaccinia virus occurs through adapter IPS-1 signaling

Abstract

Interferon regulatory factor 3 (IRF-3) undergoes phosphorylation-induced activation in virus-infected cells and plays an important role in the antiviral innate immune response. The E3L protein encoded by vaccinia virus is known to impair phosphorylation and activation of IRF-3. Kinases in addition to I kappaB kinase-related kinases are implicated in the IRF-3-dependent antiviral response. To test in human cells the role of the protein kinase regulated by RNA (PKR) in IRF-3 activation, HeLa cells made stably deficient in PKR using an RNA interference strategy were compared with PKR-sufficient cells. Rapid phosphorylation and nuclear accumulation of IRF-3 were detected in PKR-sufficient cells following infection with E3L deletion mutant (DeltaE3L) virus. By contrast, the full IRF-3 activation response was largely abolished in PKR-deficient cells. The DeltaE3L virus-induced IRF-3 activation seen in PKR-sufficient cells was diminished by treatment with cytosine beta-D-arabinofuranoside. Furthermore, the vaccinia mutant ts23, which displays increased viral double-stranded RNA production at 39 degrees C, induced PKR-dependent IRF-3 phosphorylation at 39 degrees C but not at 31 degrees C. Both IRF-3 phosphorylation and cell apoptosis induced by infection with DeltaE3L virus were dependent upon RIG-I-like receptor signal transduction components, including the adapter IPS-1. These data suggest that PKR facilitates the host innate immune response and apoptosis in virus-infected cells by mediating IRF-3 activation through the mitochondrial IPS-1 signal transduction pathway.

FIGURE 1.

PKR-dependent phosphorylation and nuclear localization of IRF-3 in vaccinia virus-infected cells is impaired by E3L. A, whole cell extracts were prepared from mock-infected cells (lanes 1, 4, and 7) or cells infected for 10 h with the either wild-type (WT)(lanes 2, 5, and 8) or the E3L deletion mutant (ΔE3L) (lanes 3, 6, and 9) vaccinia virus and analyzed by Western immunoblot assay with antibody against IRF-3 and PKR. β-Actin was used as a loading control. Cells used were as follows: PKR-sufficient parental (PKR⁺) and knockdown control (PKR^kd-con); PKR-deficient knockdown (PKR^kd). B, subcellular distribution of IRF-3. Cells were mock-infected or infected with either wild-type (WT) or ΔE3L mutant virus and analyzed for IRF-3 subcellular localization at 8 h after infection by indirect immunofluorescence. C, time course of IRF-3 phosphorylation in parental PKR⁺, PKR-deficient knockdown PKR^kd, and PKR-sufficient knockdown control PKR^kd-con cells. Cells were mock-infected (0) or infected for 3, 6, 9, or 12 h with either WT (left) or ΔE3L mutant (right) virus, and whole cell extracts were prepared, and immunoblot analyses were performed with antibodies against IRF-3 and α-tubulin as a loading control.

FIGURE 2.

Ara C blocks PKR-dependent phosphorylation in ΔE3L mutant virus-infected cells. Whole cell extracts were prepared from uninfected (–) or ΔE3L virus-infected (+) cells at 10 h after infection, either untreated (–) or treated (+) with Ara C, and analyzed by Western immunoblot assay with antibody against IRF-3. α-Tubulin was used as a loading control. Cells used were as follows: PKR-sufficient parental (PKR⁺) and knockdown control (PKR^kd-con); PKR-deficient knockdown (PKR^kd).

FIGURE 3.

PKR-dependent IRF-3 phosphorylation and inhibition of viral capsid protein expression displays a temperature-sensitive phenotype in ts23 mutant vaccinia virus-infected cells. Whole cell extracts were prepared from mock-infected (M) cells or cells infected with either WT, ΔE3L mutant (ΔE), or with ts23 mutant (ts) vaccinia virus at 31 °C (A) or 39 °C (B). Cell used were as follows: PKR-sufficient parental (PKR⁺) and knockdown control (PKR^kd-con); PKR-deficient knockdown (PKR^kd). Western immunoblot analyses were carried out with antibodies against IRF-3, PARP, and vaccinia virus proteins, and α-tubulin as a loading control. PARP 116, intact PARP; PARP 85, cleavage product.

FIGURE 4.

IRF-3 phosphorylation following infection of PKR⁺, PKR^kd, and PKR^kd-con with mutant viruses expressing truncated E3L proteins lacking the ZBM or RBM domain. Whole cell extracts were prepared from uninfected cells (Mock) or infected cells at 10 h post-infection with either WT or the following E3L deletion mutant viruses: ΔE3L deletion mutant; Δ26C mutant lacking RBM; Δ83N mutant lacking ZBM. Cells used were as follows: PKR-sufficient parental (PKR⁺) and knockdown control (PKR^kd-con); PKR-deficient knockdown (PKR^kd). Western immunoblot analysis was carried out with antibody against IRF-3, PKR, and β-actin as a loading control.

FIGURE 5.

PKR-mediated IRF-3 phosphorylation occurs within the RIG-like receptor-IPS adapter signal transduction pathway. A, whole cell extracts were prepared from parental PKR⁺ cells, either uninfected (–) or ΔE3L virus-infected (+) for 10 h following transient knockdown as described under “Experimental Procedures,” utilizing chemically synthesized siRNAs against luciferase as a control (lanes 1 and 2), IPS-1 (lanes 3 and 4), or TRIF (lanes 5 and 6). Western immunoblot analyses were carried out with antibodies against IRF-3, phospho-IRF-3(Ser-396), TRIF, IPS-1, and β-actin as a loading control. B, whole cell extracts were prepared from parental PKR⁺ cells infected with ΔE3L mutant virus following transient knockdown utilizing siRNAs against the following targets: lane 1, luciferase control; lane 2, RIG-I; lane 3, mda-5; lane 4, IPS-1; lane 5, IRF-3; lane 6, PKR; lane 7, RIG-I and mda-5. Western immunoblot analyses were carried out with extracts prepared at 10 h after infection with antibodies against IRF-3, IPS-1, phospho-PKR (P-PKR), PKR, phospho-eIF-2α (P-eIF-2α), and eIF-2α or 24 h after infection to measure PARP cleavage (PARP 85), vaccinia virus protein expression, and β-actin.