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.
Highlights
Stranded RNA act through adapter proteins, TIR domaincontaining adapter-inducing IFN- (TRIF) for the endosome membrane receptor TLR3 and IFN--promoter simulator 1 (IPS-1, named as VISA, MAVS) for the retinoic acid-inducible gene (RIG)-I and mda-5 RLRs, to induce the synthesis of type I interferons (IFNs) [2,3,4]
Production of dsRNA occurs during the life cycle of several viruses, both RNA viruses and DNA viruses [13, 14]. dsRNA acts as an effector of multiple cellular enzymes that are part of the IFN response, including the protein kinase regulated by dsRNA (PKR) [4, 15,16,17]; the family of dsRNA-dependent 2Ј,5Ј-oligoadenylate synthetases [4, 17]; and ADAR1 [17, 18]
PKR Mediates interferon regulatory factor 3 (IRF-3) Phosphorylation through an IPSdependent Signaling Pathway—To test whether PKR mediates IRF-3 activation in response to infection with the ⌬E3L mutant Vaccinia virus (VV) through the RIG-I-like receptor signal transduction pathway or the TLR3 receptor that senses dsRNA [4], we examined the effect of transient knockdown of the cognate adapter proteins, IPS-1 for the RIG-I-like receptors, and TRIF for TLR3
Summary
Cells and Viruses—HeLa, baby hamster kidney, and RK13 cells were maintained in Dulbecco’s modified Eagle’s medium complemented with 5 or 10% (v/v) fetal bovine serum (Hyclone), respectively, 100 g/ml penicillin, and 100 units/ml streptomycin (Invitrogen) as described previously [36]. 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 (⌬E3L) (lanes 3, 6, and 9) vaccinia virus and analyzed by Western immunoblot assay with antibody against IRF-3 and PKR. Cells were mock-infected (0) or infected for 3, 6, 9, or 12 h with either WT (left) or ⌬E3L mutant (right) virus, and whole cell extracts were prepared, and immunoblot analyses were performed with antibodies against IRF-3 and ␣-tubulin as a loading control. In cells infected with the ⌬E3L mutant, two additional IRF-3 forms, corresponding to the C-terminal phosphorylation of IRF-3 [41, 42], were seen in the two PKR-sufficient cell lines (PKRϩ and PKRkd-con) (Fig. 1A, lanes 3 and Indirect Immunofluorescence Microscopy—Cells were seeded 9). Coverslips were mounted Dependent upon PKR in ⌬E3L Mutant-infected Cells—The using ProLong Gold antifade reagent with 4Ј,6-diamidino-2- phosphorylation of IRF-3 on Ser and Thr in the C-terminal phenylindole (Invitrogen); stained samples were visualized region, which corresponds to the most slowly migrating forms using an Olympus fluorescence microscope
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