Identification of a novel pathway essential for the immediate-early, interferon-independent antiviral response to enveloped virions

Abstract

Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.

FIG. 1.

Inhibition of the PI3 kinase and SAPK/JNK pathways, but not the ERK 1/2 and p38 MAPK pathways, prevents the induction of ISG56 mRNA in response to enveloped virus particles. HEL fibroblasts were mock infected or treated with the indicated stimuli. At 6 h postinfection, whole-cell extracts were harvested to investigate the level of protein kinase phosphorylation. At 12 h postinfection, RNA was harvested to investigate the level of ISG56 mRNA accumulation. HEL fibroblasts were treated with the above stimuli in the presence or absence of the following: A, U0126 (10 μM); B, SB202190 (25 μM); C, SP600125 (25 μM); D, LY294002 (50 μM). To ensure maintenance of inhibitor activity, each positive control was administered 30 min prior to harvest. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 2.

Entry of HSV-1 and HCMV into human fibroblasts is not prevented in the presence of the LY294002 and SP600125 inhibitors. HEL fibroblasts were treated with HSV (A) or HCMV (B) at an MOI of 10 PFU/cell in the presence of DMSO, LY294002 (50 μM), or SP600125 (25 μM). At the indicated times postinfection, cells were fixed and stained for HSV-1- or HCMV-specific viral protein expression. Cells positive for viral protein were counted and expressed as a percentage of the total number of cells from 12 random fields of view from three independent experiments. Statistical analysis was performed with a t test. Where applicable, a statistical significance of P = 0.01 is noted (*).

FIG. 3.

The prototypic PI3 kinase, PI3K, and its downstream effector, Akt/PKB, are not essential for the induction of ISG56 protein in response to enveloped virus particles. HEL fibroblasts were preloaded with either AdE1/E3 or Addnp85 for 24 h. Cells were then serum starved for 2 h, including a 1-h pretreatment with LY294002 where applicable. After a further 24-h treatment, whole-cell extracts were harvested and Western blot analysis was performed. To ensure maintenance of inhibitor activity, 25 ng/ml of PDGF was added 30 min prior to harvest. Twenty-five micrograms of total protein was loaded onto a 7.5% sodium dodecyl sulfate-polyacrylamide gel.

FIG. 4.

DNA-PK activity is not required for the induction of ISG56 mRNA in response to enveloped virus particles. DNA-PKcs^+/− and DNA-PKcs^−/− primary MEFs were pretreated with 50 μg/ml cycloheximide or DMSO for 30 min. Cells were then infected with HSV or HCMV in the presence or absence of cycloheximide for 6 h, at which time RNA was harvested and ISG56 levels detected by RT-PCR analysis. An asterisk indicates the presence of IFN in supernatants as determined by standard plaque reduction assays on naive cells. CHX, cycloheximide.

FIG. 5.

Inhibition of PAK activity does not prevent the induction of ISG15 and ISG56 proteins in response to virus particle stimulation. A549 epithelial cells were preloaded with AdE1/E3 or AdPAKK299R for 24 h. (A) Immunofluorescence analysis of dominant negative PAK expression. Scale bar = 95 μm. (B) Morphological analysis of dominant negative PAK expression. Arrowheads indicate the perimeter of the cell expressing GFP-tagged dominant negative PAK. Scale bar = 25 μm. (C) Western blot analysis of PAK (5 μg extract), ISG15, and ISG56 (20 μg extract) protein levels following infection with AdPAKK299R.

FIG. 6.

Treatment with the LY294002 inhibitor does not prevent the nuclear translocation of IRF-3 in response to enveloped virus particles. (A and B) HEL fibroblasts were treated with HSV-UV (MOI, 10 PFU/cell) in the presence or absence of 50 μM of LY294002. (C and D) HEL fibroblasts were treated with HCMV-UV (MOI, 10 PFU/cell) in the presence or absence of 50 μM LY294002. Cells were fixed and stained, and nuclei positive for IRF-3 expression were counted at various times postinfection as described in Materials and Methods. Statistical analysis was performed by a one-way analysis of variance with the Tukey test post hoc. Statistically significant differences (P < 0.001) are noted (**).

FIG. 7.

Treatment with the LY294002 inhibitor does not prevent dimerization of IRF-3 in response to enveloped virus particles. HEL fibroblasts were treated with HCMV-UV (MOI, 10 PFU/cell) in the presence and absence of the LY294002 inhibitor. Eight micrograms of cytoplasm-enriched (C) and 2 μg of nuclear (N) extracts were separated by native polyacrylamide gel electrophoresis, followed by Western blot analysis with an antibody specific to the native conformation of IRF-3.