A novel inhibitor of the NF-{kappa}B signaling pathway encoded by the parapoxvirus orf virus

Abstract

The parapoxvirus orf virus (ORFV) is a pathogen of sheep and goats that has been used as a preventive and therapeutic immunomodulatory agent in several animal species. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate and manipulate immune responses are poorly understood. Here, the novel ORFV protein ORFV024 was shown to inhibit activation of the NF-kappaB signaling pathway, an important modulator of early immune responses against viral infections. Infection of primary ovine cells with an ORFV024 deletion mutant virus resulted in a marked increase in expression of NF-kappaB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-alpha-induced phosphorylation and nuclear translocation of NF-kappaB-p65, phosphorylation, and degradation of IkappaBalpha, and phosphorylation of IkappaB kinase (IKK) subunits IKKalpha and IKKbeta, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-kappaB activating stimuli. Although ORFV024 interferes with activation of the NF-kappaB signaling pathway, its deletion from the OV-IA82 genome had no significant effect on disease severity, progression, and time to resolution in sheep, indicating that ORFV024 is not essential for virus virulence in the natural host. This represents the first description of a NF-kappaB inhibitor encoded by a parapoxvirus.

FIG. 1.

Transcription kinetics and subcellular localization of ORFV024. (A) Transcription kinetics of ORFV024, ORFV127, and ORFV055 during ORFV infection cycle in OFTu cells in the presence or absence of AraC as determined by RT-PCR. (B) ORFV024 punctate localization in the cytoplasm of 024GFP/HeLa stable cell lines.

FIG. 2.

Growth characteristics of ORFV024 deletion mutant virus OV-IA82Δ024. (A) Multiple step growth curves of wild-type and mutant virus in primary OFTu cells. One-step growth curves of wild-type and mutant virus in primary OFTu (B) and in HeLa cells (C). (D) CPE of wild-type and mutant virus in primary OFTu cells 48 h after infection (MOI = 10) (magnification, ×20). (E) Plaque morphology of wild-type and mutant viruses in OFTu cells. At 72 h after infection cells were fixed with formaldehyde (3.7%) and stained with 1% crystal violet (magnification, ×20). Black bars demarcate plaques produced by OV-IA82 and OV-IA82Δ024.

FIG. 3.

Effect of ORFV024 on NF-κB-mediated transcription. (A) Primary OFTu cells were cotransfected with a vector encoding a firefly luciferase gene under the control of NF-κB (pNFκB-Luc) and with a plasmid encoding sea pansy (Renilla reniformis) luciferase under the control of herpesvirus TK promoter (pRL-TK). At 24 h after transfection cells were mock infected or infected with OV-IA82, or OV-IA82Δ024. Firefly and sea pansy luciferase activities were measured at 2, 4, and 6 h p.i. and expressed as relative fold changes in luciferase activity (*, P < 0.05; **, P < 0.005). (B) OFTu cells were cotransfected with pNFκB-Luc, pRL-TK, and pCMV024-Flag. At 24 h after transfection the cells were exposed to MEM containing 2% FBS and 0 or 250 ng of LPS/ml for 6 h. Cells were harvested, and firefly and sea pansy luciferase activities were measured and expressed as fold changes in luciferase activity (‡, P < 0.01). (C) Western blot analysis of cell lysates from samples tested in B (anti-Flag M2 antibody). (D) HeLa cells stably expressing GFP or ORFV024-GFP were cotransfected with pNFκB-Luc and pRL-TK. At 24 h after transfection, cells were exposed to MEM containing 2% FBS and 0, 20, or 50 ng of TNF-α/ml for 6 h. Cells were harvested, and the firefly and sea pansy luciferase activities were determined. Firefly luciferase activity was normalized to sea pansy luciferase activity and fold changes were calculated (*, P < 0.05). (E) Western blot analysis of cell lysates representative of samples tested in panel D (anti-GFP antibody). The results are representative of two (A and B) or three (D) independent experiments.

FIG. 4.

Effect of ORFV024 expression on NF-κB-p65 phosphorylation and nuclear translocation. (A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) were treated with TNF-α (20 ng/ml) and harvested at the indicated times (UN, untreated controls). Fifty micrograms of total protein extracts were resolved by SDS-PAGE, blotted, and probed with antibodies against proteins indicated on the right. (B) Densitometry of phosphor NF-κB-p65 bands normalized to loading control Gβ (*, P < 0.05; **, P < 0.005). (C) OFTu cells were transfected with vectors pCMVTag4A (empty) or pCMV024-Flag, treated with TNF-α (20 ng/ml), and harvested at the indicated times (UN, untreated controls). Fifty micrograms of total protein extracts were resolved by SDS-PAGE, blotted, and probed with the antibodies directed against the proteins indicated on the right. (D) Densitometry of phosphor NF-κB-p65 bands normalized to loading control Gβ (*, P < 0.05). (E) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) were treated with TNF-α (20 ng/ml) for 45 min, and cytoplasmic and nuclear protein fractions were extracted (UN, untreated controls). Twenty micrograms of total protein extracts were resolved by SDS-PAGE, blotted, and probed with antibodies against NF-κB-p65 (top panels), GAPDH (bottom left panel), or histone H3 (bottom right panel). The results are representative of two (E) or three independent experiments (A, B, C, and D).

FIG. 5.

Effect of ORFV024 expression on IκBα phosphorylation and degradation. (A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) fusion protein were treated with TNF-α (20 ng/ml) and harvested at the indicated times (UN, untreated controls). Fifty micrograms of total protein extracts were resolved SDS-PAGE, blotted, and probed with the antibodies directed against the proteins indicated on the right. (B) Densitometry of phosphor IκBα bands normalized to Gβ (*, P < 0.005). The results are representative of five independent experiments.

FIG. 6.

Effect of ORFV024 expression on IKKα and IKKβ phosphorylation. HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) fusion protein were treated with TNF-α (20 ng/ml) and harvested at the indicated times (UN, untreated controls). Fifty micrograms of total protein extracts were resolved by SDS-PAGE, blotted, and probed with the antibodies against the proteins indicated on the right. The results are representative of three independent experiments.

FIG. 7.

Clinical course of orf in sheep. Groups of four- to five-month-old lambs were inoculated with MEM, OV-IA82, or OV-IA82Δ024 at the mucocutaneous border of the lower lip following scarification. Characteristic orf lesions (arrows) were observed in both OV-IA82 and OV-IA82Δ024 inoculated lambs (days 3, 7, and 12 p.i.).