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J Virol. 2010 April; 84(8): 3962–3973.
Published online 2010 February 10. doi:  10.1128/JVI.02291-09
PMCID: PMC2849485

A Novel Inhibitor of the NF-κB Signaling Pathway Encoded by the Parapoxvirus Orf Virus[down-pointing small open triangle]


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-κB 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-κB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-α)-induced NF-κB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-α-induced phosphorylation and nuclear translocation of NF-κB-p65, phosphorylation, and degradation of IκBα, and phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-κB activating stimuli. Although ORFV024 interferes with activation of the NF-κB 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-κB inhibitor encoded by a parapoxvirus.

Orf virus (ORFV) is the type member of Parapoxvirus, one of the eight genera of the subfamily Chordopoxvirinae of the Poxviridae (43). Other members of the genus include bovine papular stomatitis virus (BPSV), pseudocowpox virus, and parapoxvirus of red deer in New Zealand (43). Parapoxviruses are characterized by the ovoid shape of virions, the presence of criss-cross-arranged surface tubulelike structures, and the high G+C content of their genomes (21).

ORFV is the causative agent of orf (contagious ecthyma, contagious pustular dermatitis, or scabby mouth), a nonsystemic, highly contagious, ubiquitous disease of sheep and goats which is characterized by maculopapular and proliferative lesions affecting the skin around the mouth, nostrils and teats, and the oral mucosa (29). Lesions are largely confined to areas surrounding the virus entry sites, evolving through stages of erythema, vesicles, pustules, and scabs (21). In the absence of secondary infections, lesions are usually resolved in 6 to 8 weeks; however, persistent infections have been reported (13, 27, 48). Orf primarily affects animals less than 1 year old, and morbidity in a susceptible flock may reach 90%. Mortality is usually low; however, lesions in lips and udders may prevent affected animals from suckling, which can result in rapid emaciation (39). In spite of a vigorous and typical antiviral T-helper type 1 (Th1) immune response, immunity elicited by ORFV is short-lived, and animals can be repeatedly infected, albeit lesions are smaller and resolve sooner than in primary infections (28, 40, 69). Orf is a zoonotic disease, affecting humans in close contact with infected animals (17, 44, 55).

The ORFV genome is approximately 138 kbp, with a G+C content of 64%, and contains 131 putative genes, 89 of which are conserved in all characterized chordopoxviruses (14, 61). Based on sequence homology to host and viral genes, several immunomodulatory genes with putative virulence functions have been identified in ORFV, including a homologue of interleukin-10 (IL-10; ORFV127), chemokine binding protein (ORFV112), inhibitor of granulocyte-macrophage colony-stimulating factor and IL-2 (GIF; ORFV117), vascular endothelial growth factor (ORFV132), and interferon (IFN) resistance gene (ORFV020) (reviewed in reference 21). Recently, screening of vaccinia virus-orf virus recombinants led to identification of a Bcl-2-like inhibitor of apoptosis (ORFV125) (66). Notably, ORFV encodes 15 mostly terminally located genes, which lack similarity to other poxvirus or cellular proteins and with putative virulence and host range functions (14).

Historically, ORFV has been used in veterinary medicine as a preventive and therapeutic immunomodulatory agent (reviewed in reference 64). Live or inactivated ORFV preparations exhibit dose-dependent immunomodulatory effects, with therapeutic efficacy and a favorable side effect profile demonstrated for various diseases, including infectious diseases of companion and farm animals (5, 7, 18, 19, 38, 70). Inactivated ORFV has been shown to induce an autoregulatory cytokine response in mice, involving upregulation of Th1-type cytokines (IL-12, IL-18, and IFN-γ) and their subsequent downregulation, which is accompanied by the induction of IL-10 and IL-4 (64). In addition, inactivated ORFV promotes activation of monocytes and dendritic cells via Toll-like receptor (TLR)-dependent and independent pathways, leading to the synthesis and release of early proinflammatory cytokines (22, 60). Currently, ORFV functions (genes, proteins, and mechanisms of action) associated with modulation and manipulation of early host immune responses are poorly understood.

Many viruses, including hepatitis C virus, paramyxoviruses, influenza virus, African swine fever virus, and poxviruses, encode for proteins that disrupt or modulate immune responses by targeting specific aspects of the nuclear factor κB (NF-κB) signaling pathway (reviewed in references 31 and 63). By binding to specific promoter sequences, NF-κB mediate expression of a wide variety of cellular genes, including many involved in innate immunity, inflammation, and apoptosis, which are critical for early antiviral responses (31, 52, 57, 63). The activity of NF-κB is regulated by its association with the inhibitory IκB molecules, which sequester NF-κB in the cytoplasm (33). Various stimuli, including the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1, bacterial lipopolysaccharide (LPS), viruses, and viral products, lead to phosphorylation of IκB proteins by IκB kinases, resulting in proteasomal degradation of IκB and nuclear translocation of NF-κB subunits (33). NF-κB subunits are subject to extensive posttranslational modifications, which may facilitate their nuclear translocation or define their transcriptional functions in the nucleus (9, 53). The critical IκB kinase (IKK) complex is the bottleneck for most NF-κB-activating pathways (33, 57), including those initiated at TLRs and TNF receptors (8), and several viruses have evolved strategies to inhibit IKK complex activation (reviewed in reference 56).

Poxviral proteins involved in inhibition of the NF-κB pathway have been identified in members of the genera Orthopoxvirus, Leporipoxvirus, Yatapoxvirus, and Molluscipoxvirus, with selected viruses encoding for multiple inhibitors [for a review, see reference 41]. For example, the type member of the Poxviridae vaccinia virus (VACV) encodes at least seven NF-κB inhibitors (A52R, A46R, B14, K1L, N1L, M2L, and E3L), which target different steps leading to NF-κB activation, most often by preventing IKK complex activation (11, 15, 24, 30, 46, 59, 62). Notably, deletion of individual genes encoding selected VACV NF-κB inhibitors from the viral genome resulted in various degrees of attenuation in mice. However, no individual gene deletion rendered complete attenuation (2, 10, 30), suggesting that encoded proteins exhibit, to some extent, complementary functions during VACV infection (41).

Homologues of known poxviral NF-κB inhibitors are absent in parapoxviruses, suggesting that either they encode novel NF-κB inhibitors or employ alternative immune evasion strategies. We present data here demonstrating that ORFV024, a gene unique to parapoxviruses, profoundly affects chemokine and other proinflammatory gene transcription in infected cells. Expression of ORFV024 in cell cultures decreased the phosphorylation and nuclear translocation of NF-κB-p65, the phosphorylation and degradation of IκBα, and the phosphorylation of IKKα and IKKβ, indicating that ORFV024 expression suppresses early cellular responses to ORFV infection by inhibiting activation of the NF-κB pathway. Notably, ORFV024 is not essential for viral virulence since its deletion did not affect ORFV pathogenesis in sheep.


Cells and virus.

Primary ovine fetal turbinate (OFTu) cells were kindly provided by Howard D. Lehmkuhl (USDA) and were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 50 μg of gentamicin/ml, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. HeLa cells (American Type Culture Collection) were maintained in Dulbecco modified essential medium supplemented with 10% FBS, 2 mM l-glutamine, 50 μg of gentamicin/ml, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. ORFV strain OV-IA82 was isolated from sheep nasal secretions at the Iowa Ram test Station during an orf outbreak in 1982, and its genome has been fully sequenced (14). Low-passage parental OV-IA82 virus was used to construct the deletion mutant virus OV-IA82Δ024 and in all procedures involving infections with wild-type virus, PCR amplification, and cloning of viral genes.


ORFV024 coding sequence was PCR amplified from the OV-IA82 genome and cloned into the expression vectors pCMVTag4A (Stratagene, La Jolla, CA) and pEGFP-N1 (Clontech, Mountain View, CA). PCR primers were 024Flag-Fw(HindIII)-5′-TAAGGCCTCTAAGCTTATGGCCTCCTACGTCAGCGGCGCT-3′, 024Flag-Rv(XhoI)-5′-CAGAATTCGCCTCGAGCACGTAGACCGTGTGCGTGCGC-3′, 024EGFP-Fw(XhoI)-5′-ATCTTAGCTCGAGCTAAGATGGCCTCCTAC-3′, and 024EGFP-Rv(HindIII)-5′-CAGAATAAGCTTCACGTAGACCGTGTGCGTG-3′. The restriction enzymes used for cloning are indicated on each primer. DNA sequencing of constructs confirmed the integrity of ORFV024 sequence and in-frame cloning of FLAG (pCMV024-Flag) and EGFP (p024EGFP) tag proteins.

For mutant virus generation, a recombination cassette was constructed by PCR amplification of ORFV024 left (LF; 856 bp) and right (RF; 866 bp) flanking regions from the OV-IA82 genome, followed by cloning of amplicons into vector pZippy-Neo/Gus (16). Primers used for amplification were 024LF-Fw(HindIII)-5′-TAAGGCCTCTAAGCTTAACCAGCAGACCTTCTTCACCAA-3′, 024LF-Rv(SalI)-5′-CAGAATTCGCGTCGACCTTAGCTCTGTCTGAACTGAAGCA-3′, 024RF-Fw(NotI)-5′-ATTCTTATGCGGCCGCGCCGGCTTCATCCGCCGCAGCATA-3′, and 024RF-Rv(BglII)-5′-CAGAATTCGCAGATCTTACGGCGACACCGACTCCGTGTTC-3′. Restriction enzymes used for cloning are indicated on each primer. The recombination cassette pZ024LF-Neo/Gus-024RF was constructed by cloning ORFV024 LF and RF flanking regions on each side of a reporter construct consisting of neomycin (Neo) resistance and β-glucuronidase (Gus) genes preceded by VACV VV7.5 and modified H5 promoters, respectively.

ORFV024 sequence alignment.

Alignment of ORFV024 was performed using CLUSTAL W (Biology Workbench). Accession numbers of the aligned proteins are AAR98119.1 (OV-IA82), ABA00541.1 (OV-NZ2), AAP76384.1 (OV-Orf11), AAR98249.1 (OV-SA00), AAR98381.1 (BPSV-AR02), and GQ911582 (BPSV- TX-09).


Transcription kinetics of ORFV024 was assessed during ORFV infection in vitro by reverse transcription-PCR (RT-PCR). OFTu cells were cultured in 35-mm dishes for 16 h and inoculated with the wild-type virus OV-IA82 (multiplicity of infection [MOI] = 10) in the presence or absence of cytosine arabinoside (AraC; 40 μg/ml; Sigma Aldrich, St. Louis, MO) an inhibitor of DNA replication and, consequently, of late poxviral transcription (12). Cells were harvested at 0, 1, 2, 3, 6, 12, and 24 h postinoculation (p.i.), and total RNA was extracted using TRIzol reagent (Invitrogen, San Diego, CA). Samples were treated with DNase I (New England Biolabs, Ipswich, MA) for 10 min at 37°C, and RNA was purified by using the RNeasy minikit (Qiagen, Valencia, CA). A total of 2 μg of RNA was reverse transcribed by using a Superscript III kit (Invitrogen), following the manufacturer's protocol. The oligo(dT) used in RT reactions was oligo-dT-5′-ATGGATGCCTACAGCGCTCTTTTTTTTTTTTTTTTTTTT-3′. ORFV024, ORFV127 (early gene control), and ORFV055 (late gene control) transcription was assessed by using 1 μl of cDNA and the following primers 024intFw-5′-GCGGACACAGCCACAACCACAGTC-3′, 024intRv-5′-CTAGCACGCGCTTTCGGTACCGCC-3′, 127EintFw-5′-CTCCTCGACGACTTCAAAGG-3′, 127EintRv-5′-TATGTCGAACTCGCTCATGG-3′, 055LFw-5′-AATCATGGATCCGCCACCATGTTCTTCGCGCGTCGC-3′, and 055LRv-5′-TATCATCTCGAGCGGGCGTGGAGGTCGCCGACC-3′. Negative controls and controls for DNA contamination (no reverse transcriptase) were included in all reactions.

Construction and characterization of ORFV024 deletion mutant virus OV-IA82Δ024.

OV-IA82Δ024 was obtained by homologous recombination between the parental ORFV strain OV-IA82 and the recombination cassette pZ024LF-Neo/Gus-024RF. OFTu cells cultured in 25-cm2 flasks were infected with OV-IA82 (MOI = 1) and 3 h later transfected with pZ024LF-Neo/Gus-024RF (10 μg) using Lipofectamine 2000 according to manufacturer's recommendations. At 48 h after infection/transfection, cultures were harvested, and cell lysates were used for selection of recombinant viruses by plaque assay. OFTu cells cultured in six-well plates were infected with serial 10-fold dilutions of cell lysates from the infection/transfection and overlaid with culture medium containing 0.5% SeaKem GTC agarose (Cambrex Bioscience, Rockland, ME) and 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc; 0.5 μg/ml, Gold Biotechnologies, St. Louis, MO). Blue plaques indicative of recombination were harvested and subjected to additional rounds of plaque purification. The absence of ORFV024 sequence and presence of Neo/Gus sequences in the purified recombinant virus were confirmed by PCR and Southern blotting. The primers used for PCR amplification of ORFV024 and Neo/Gus reporter gene were 024intFw-5′-GCGGACACAGCCACAACCACAGTC-3′, 024intRv-5′-CTAGCACGCGCTTTCGGTACCGCC-3′, NG2Fw-5′-ATCAGGACATAGCGTTGGCTACC-3′, and NG2 Rv-5′-TGCCGTAATGAGTGACCGCATCGA-3′, respectively. To assess the integrity of regions involved in recombination, 824 and 874 bp on the left and right flanking regions of ORFV024 were sequenced by using an Applied Biosystems Prism 3730 X1 automated DNA sequencer (Applied Biosystems, Foster City, CA).

Growth characteristics of deletion mutant virus OV-IA82Δ024 and wild-type virus OV-IA82 were analyzed and compared in primary OFTu cells and in HeLa cells. Multiple step growth curve was performed in OFTu cells inoculated with mutant or wild-type virus (MOI = 0.1), and at 12, 24, 48, and 72 h postinfection (p.i.) viral yields were quantitated in cultures of OFTu cells by the Spearman-Karber's 50% tissue culture infectious dose (TCID50) method. One-step growth curves were performed in OFTu and HeLa cells inoculated with mutant or wild-type virus (MOI = 10), and at 6, 12, 24, and 48 h p.i. viral yields were quantitated in cultures of OFTu cells as described above. To compare the cytopathic effect (CPE) induced by OV-IA82Δ024 and OV-IA82, cells were inoculated with each virus (MOI = 10) and 48 h p.i. evaluated under an inverted light microscope (Leica DMI 4000 B; 20×). To analyze plaque sizes and morphology, OFTu cells were cultured in six-well plates inoculated with serial 10-fold dilutions of each virus (10−4 to 10−6) and fixed with 3.7% formaldehyde, 72 h p.i. Cells were stained with 1% crystal violet for 15 min at room temperature, and plaques were examined under an inverted microscope (Leica DMI 4000 B; 20×).

Microarray and real-time PCR analysis.

Gene expression profile of virus-infected OFTu cells was initially analyzed by microarray and then confirmed by real-time PCR. OFTu cells grown in 35 mm dishes for 16 h at 37°C with 5% of CO2 were inoculated with wild-type or mutant virus (MOI = 10) and harvested for total RNA extraction with TRIzol reagent (Invitrogen, San Diego, CA) at 2 and 4 h p.i.

For microarray analysis, RNA samples were processed and prepared according to standard Affymetrix protocols (Santa Clara, CA) at the Biotechnology Center of the University of Illinois at Urbana-Champaign. The gene expression profile at each time point was measured by using the Affymetrix GeneChip Bovine Genome Array which contains 24,128 probe sets that measure over 23,000 bovine transcripts. After the data passed the Affymetrix's recommended quality control, arrays were preprocessed by using the GCRMA algorithm (68) as part of the Affymetrix (23) and GCRMA (68) packages from the Bioconductor project (25). Fold changes in gene expression were calculated for each time point by comparing expression in cells infected with the mutant virus with those infected by wild-type virus.

Real-time PCR was performed to validate the microarray results. One microgram of total RNA, obtained from samples of three independent experiments, was used for cDNA synthesis as described above. Expression of genes CCL20, CXCL3, IL-1α, IL-6, IL-8, ICAM-1, IRF-1, NFκBIA, and PTGS2 was analyzed in an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). Primers and probes used to detect the expression of these genes were synthesized by Applied Biosystems (TaqMan gene expression custom assays), based on ovine gene sequences in GenBank. Real-time PCR amplifications were performed in 10-μl reactions, with 4.5 μl of cDNA (1:80 dilution in diethyl pyrocarbonate-H2O), 5 μl of TaqMan Master mix (Applied Biosystems), and 0.5 μl of the mixture of primers and probe (TaqMan gene expression custom assays). PCR conditions consisted of initial denaturation and DNA polymerase activation (AmpliTaq Gold, Applied Biosystems) at 95°C for 15 s, followed by annealing and extension at 60°C for 1 min.

Each sample was tested in triplicate and the average of the three replicates was used to calculate fold changes. Expression levels of the genes tested were normalized to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The measurement of gene expression was performed by using the relative quantitation method (67). Standard curves were prepared from a pool of cDNA (2-fold dilutions; 1:20 to 1:2,560) for GAPDH and all tested genes. Samples from mock-infected cells collected at 2 and 4 h p.i. were used as calibrators to calculate the fold changes. Negative controls (no template) and controls for DNA contamination (no RT control) were included in all reactions. Statistical analysis of the real-time PCR data was performed by using a Student t test.

Establishment of cell lines stably expressing ORFV024.

HeLa cells constitutively expressing green fluorescent protein (GFP) or ORFV024-GFP fusion protein were obtained by using a retroviral expression system (pLNCX2; Clontech). DNA sequences encoding GFP or ORFV024-GFP were cleaved from vectors pEGFP-N1 and p024EGFP with BglII and NotI and subcloned into plasmid pLNCX2. Plasmid constructs were transfected into the packaging cell lines GP2-293 using Lipofectamine 2000. At 48 h after transfection, the supernatant containing GFP- or ORFV024-GFP-encoding recombinant retrovirus particles was harvested and used to infect HeLa cells. Individual cell clones were selected, amplified, and maintained in the presence of G418 (500 μg/ml; Gibco). The expression of GFP and ORFV024-GFP was monitored by fluorescence microscopy and by Western immunoblot with an anti-GFP antibody (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA).

NF-κB luciferase reporter assays.

The ability of ORFV024 to inhibit NF-κB-transcriptional activity was investigated in vitro using a luciferase reporter assay. OFTu cells were cultured in 12-well plates (1.2 × 105 cells per well) and cotransfected the next day with the vectors pNF-κBLuc (450 ng; Clontech, Mountain View, CA) and pRLTK (50 ng; Promega, Madison, WI), which encode the firefly luciferase gene under the control of κB-responsive elements and sea pansy (Renilla reniformis) luciferase, respectively, using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, cells were mock infected or infected with wild-type (OV-IA82) or mutant virus (OV-IA82Δ024) (MOI = 10). Cells were harvested with passive lysis buffer at 2, 4, and 6 h p.i. (PLB; Promega), and luciferase activities were determined using a dual luciferase reporter assay (Promega) and a luminometer (Victor2; Perkin-Elmer, Waltham, MA).

OFTu cells transiently expressing or HeLa cells stably expressing ORFV024 were cotransfected with the reporter plasmids, treated with LPS or TNF-α, respectively, and assayed for firefly and sea pansy luciferase activities. OFTu cells cultured in 12-well plates (1.2 × 105 cells per well) were cotransfected with pNF-κBLuc (450 ng), pRLTK (50 ng), and pCMV024-Flag (500 ng), or control empty vector pCMVTag4A (500 ng), using Lipofectamine 2000. At 24 h after transfection, cells were exposed to control media or media containing LPS (250 ng/ml; Invivogen, San Diego, CA) for 6 h. LPS-treated and untreated cells were harvested with PLB, and luciferase activity was determined as described above. HeLa cells stably expressing GFP or ORFV024-GFP fusion protein were cultured in 12-well plates (1.1 × 105 cells per well) and cotransfected with pNF-κBLuc (450 ng) and pRLTK (50 ng) as described above. At 24 h after transfection, cells were exposed to control media or media containing 20 or 50 ng of TNF-α (Cell Signaling, Danvers, MA)/ml for 6 h and assayed for firefly and sea pansy luciferase activities as described above. All transfections were performed in triplicate. Luciferase activities were measured as relative light units (RLU), and firefly luciferase was normalized to the sea pansy luciferase activity to correct for transfection efficiencies. Resultant ratios were used to calculate fold changes in the luciferase activity between control and treated cells. Statistical analysis of the data was performed by using Student's t test.

Western blots.

Western immunoblots were used to assess the effect of ORFV024 expression on the NF-κB pathway. HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) were cultured in six-well plates for 48 h, treated with TNF-α (20 ng/ml, Cell Signaling), harvested at various times after TNF-α-treatment (at 5, 10, and 20 min or at 10 and 20 min), and lysed with ProteoJET mammalian lysis buffer (Fermentas, Glen Burnie, MD) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). OFTu cells cultured in six-well plates were transfected with pCMVTag4A (2 μg; control) or with pCMV024-Flag (2 μg) and, 24 h after transfection, treated with TNF-α (20 ng/ml; Cell Signaling), and harvested at 10 and 20 min after TNF-α treatment as described above. Untreated cells were used as controls in all experiments. Approximately 50 μg of total protein lysate were resolved by SDS-PAGE in 10% acrylamide gels, followed by blotting to nitrocellulose membranes (Bio-Rad). Blots were incubated overnight at 4°C with antibodies against IKKα (sc-52932; Santa Cruz), IKKβ (catalog no. 2370; Cell Signaling), phospho-IKKα/β (catalog no. 2681; Cell Signaling), IκBα (sc-371; Santa Cruz), phospho-IκBα (Ser32/36) (catalog no. 9246; Cell Signaling), NF-κB-p65 (sc-7151; Santa Cruz), phospho-NF-κB-p65 (Ser536) (catalog no. 3033; Cell Signaling), Gβ (sc-378; Santa Cruz), GAPDH (sc-25778; Santa Cruz), Flag-M2 (catalog no. 200471; Stratagene), or GFP (sc-8334; Santa Cruz). After washing to remove unbound antibodies (three times for 10 min with TBS-Tween 20 0.1%), blots were incubated with secondary goat anti-rabbit IgG-(HRP) (sc-2004; Santa Cruz) or goat anti-mouse IgG-(HRP) (catalog no. 7076; Cell Signaling) for 1.5 h at room temperature and developed using chemiluminescent reagents (ECL; Pierce-Thermo Scientific, Rockford, IL). Densitometric analysis of the blots was performed by using the software ImageJ, version 1.62 (National Institutes of Health, Bethesda, MD). Statistical analysis of the densitometry data was performed by using Student t test.

Nuclear translocation of NF-κB-p65.

Nuclear translocation of NF-κB-p65 was investigated in cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa). Cells were cultured in six-well plates for 48 h (5 × 105 cells/well), treated with TNF-α (20 ng/ml) for 45 min and harvested in phosphate-buffered saline (PBS; 0.5 ml/well). Cytoplasmic and nuclear protein fractions were extracted by using a ProteoJet cytoplasmic and nuclear protein extraction kit (Fermentas, Glen Burnie, MD), after the manufacturer's protocol. Levels of translocated NF-κB-p65 were determined by western immunoblots as described above. A total of 20 μg of cytoplasmic and nuclear protein extracts were resolved by SDS-PAGE and blotted to nitrocellulose membranes. Blots were incubated with antibodies against NF-κB-p65 (sc-7151; Santa Cruz), GAPDH (sc-25778; Santa Cruz), or histone H3 (sc-10809; Santa Cruz) and detected with a secondary goat anti-rabbit-IgG-HRP antibody, followed by incubation with enhanced chemiluminescence (ECL) substrate.

Animal inoculation.

Four to five-month-old lambs (70 to 80 lbs) were obtained from an orf-free flock and randomly allocated to three groups, consisting of mock-infected (MEM; n = 3), OV-IA82-infected (n = 3), and OV-IA82Δ024-infected lambs (n = 3). Lambs were tranquilized with xylazine (Rompun; Bayer), and sites of inoculation were cleaned with sterile water. Inoculation was performed by scarification of the mucocutaneous junction of the inferior lip, and the left axillary skin, followed by local application of 0.5 ml of virus suspension containing 107.5 TCID50/ml in MEM. Animals were monitored daily for 19 days for changes in body temperature and presence of mucocutaneous lesions, including erythema, papules, vesicles, pustules, and scabs. Skin biopsies were collected from the axilla on days 3, 5, 7, and 19 p.i. and processed for histological examination according to standard procedures. All animal procedures were reviewed and approved by the University of Nebraska-Lincoln Institutional Animal Care and Use Committee (protocol 08-01-001D of 01/22/08).


Parapoxvirus ORF024 encodes for a novel protein with no homology to other viral or cellular proteins.

ORFV and BPSV ORF024 are 288 to 292 amino acids and 227 amino acids in length, respectively, with predicted molecular masses ranging from 26.2 to 32.7 kDa. ORFV strain OV-IA82 ORFV024 shares 93 to 98%, 92%, and 62% amino acid identity with homologues from the sheep isolated ORFV strains OV-NZ2 and OV-Orf11, the goat isolated ORFV strain OV-SA00, and the BPSV strains BV-AR02 and BV-TX09, respectively. No homologues of ORFV024 were detected outside the parapoxviruses. Notably, whereas retaining residues 1 to 4 and residues 12 to 19, BPSV ORF024 lacks 64 residues present in the amino terminus of all ORFV strains. Although ORF024 carboxyl terminus is highly conserved among ORFV and BPSV strains, some degree of interspecies variability was observed at amino acid residues 116 to 132, 193 to 201, and 274 to 283. No motifs indicative of protein function were detected in ORFV024.

ORFV024 is transcribed as an early gene and the protein localizes to the cell cytoplasm.

To determine the kinetics of ORFV024 transcription, primary OFTu cells were inoculated with OV-IA82 in the presence or absence of AraC and cells were harvested at various times p.i. Transcription of ORFV024 was detected by RT-PCR as early as 1 h p.i., and the levels of expression increased thereafter during the infection cycle (up to 24 h p.i.) either in the presence or absence of AraC (Fig. (Fig.1A).1A). A similar transcription pattern was observed for the early ORFV gene ORFV127 (Fig. (Fig.1A)1A) (20). In contrast, transcription of ORFV055, which encodes for a late virion membrane protein (14) was detected at late times p.i. but was markedly decreased in AraC-treated cells (Fig. (Fig.1A).1A). These results indicate that ORFV024 belongs to the early class of poxviral genes, which is known to encode for proteins involved in immune evasion and immunomodulation (1).

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 ...

The subcellular localization of ORFV024 was investigated in HeLa cells stably expressing ORFV024-GFP fusion protein. ORFV024 localized to the cell cytoplasm, exhibiting a punctate distribution pattern in this compartment (Fig. (Fig.1B).1B). A similar distribution pattern of ORFV024-GFP was observed in stable cell lines infected with OV-IA82Δ024 (data not shown). No colocalization was detected when 024GFP/HeLa cells were transfected with plasmids encoding for markers for the nucleus (pDSRed-Nuc) (Fig. (Fig.1B),1B), endoplasmic reticulum (pDSRed-ER), mitochondria (pDSRed-Mito), or peroxisomes (pDSRed-Peroxi) (data not shown). ORFV024 was similarly distributed in OFTu cells transiently expressing ORFV024-GFP fusion protein (data not shown).

Construction and characterization of ORFV024 deletion mutant virus OV-IA82Δ024.

Using homologous recombination, ORFV024 was deleted from the OV-IA82 genome and recombinant viruses were selected, and plaque purified in the presence of X-Gluc. Deleted gene sequences were not detected in samples from cells infected with recombinant virus. In contrast, β-glucuronidase sequences were detected in DNA samples from cells infected with recombinant but not wild-type virus (data not shown). DNA sequencing of regions flanking the deleted gene confirmed the integrity of parental virus sequences in the mutant virus (data not shown).

ORFV024 is nonessential for ORFV replication in vitro but affects CPE and plaque morphology.

Replication properties of OV-IA82Δ024 and OV-IA82 were compared after infection of primary OFTu cells and HeLa cells. No significant differences in replication kinetics and viral yields were detected between wild-type and mutant virus either in OFTu or in HeLa cells, indicating that ORFV024 is nonessential for ORFV replication in these cells (Fig. 2A, B, and C). Cytopathic effect (CPE) and plaque morphology induced by wild-type and mutant virus differed in OFTu-infected cells. While CPE induced by OV-IA82 was characterized by enlargement and flattening of infected cells, followed by cell rounding (Fig. (Fig.2D),2D), CPE induced by OV-IA82Δ024 was characterized by cell rounding, followed by arrangement of cells in grapelike clusters, and absence of enlarged flattened cells (Fig. (Fig.2D).2D). Although the plaques produced by wild-type and mutant virus were of similar size, OV-IA82Δ024 plaques exhibited a distinct morphology (Fig. (Fig.2E).2E). OV-IA82 plaques consisted of large flat cells around a small cell-free central area, whereas OV-IA82Δ024 plaques were characterized by a large cell-free central area and absence of flattened cells (Fig. (Fig.2E2E).

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 ...

OV-IA82Δ024 infection results in an increased expression of NF-κB-regulated genes in primary OFTu cells.

To investigate potential function(s) for ORFV024 during ORFV infection, microarray analysis was performed to determine the profile of gene expression in OFTu cells infected with wild-type or ORFV024 deletion mutant virus. Cells infected with OV-IA82Δ024 exhibited a marked increase in expression of some chemokines and other proinflammatory genes (Table (Table1),1), most of which are known to be regulated by the NF-κB family of transcription factors (51). Notably, genes CCL20, CXCL1, CXCL2, IL-6, IL-8, NFκBIA, and PTGS2 were markedly upregulated (fold increases up to 205.1, 73.7, 69.9, 8.9, 11.4, 9.1, and 9.3, respectively) in OV-IA82Δ024-infected cells at 2 and 4 h p.i. (Table (Table1).1). The microarray data set was deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE19415.

Summary of NF-κB-regulated genes detected by microarray and real-time PCR in primary OFTu cells infected with OV-IA82 or ORFV024 deletion mutant virus OV-IA82Δ024

Microarray data were confirmed with real-time PCR, with all genes tested exhibiting increased expression in OV-IA82Δ024-infected cells (Table (Table1).1). No significant differences were observed between mock-infected and OV-IA82-infected cells (Table (Table1).1). These results indicate that expression of ORFV024 inhibits the transcription of NF-κB-regulated genes in wild-type virus-infected cells, likely by inhibiting the NF-κB signaling pathway.

Deletion of ORFV024 from the OV-IA82 genome results in increased NF-κB-mediated gene transcription in infected cells.

To investigate the effect of ORFV024 on NF-κB transcriptional activity, primary OFTu cells were transfected with a plasmid encoding a luciferase reporter gene under the control of a NF-κB-responsive promoter and subsequently inoculated with OV-IA82 or OV-IA82Δ024 or mock infected. Luciferase activity levels as determined at 2, 4, and 6 h p.i. were similarly low in mock-infected and in wild-type virus-infected cells. In contrast, infection with OV-IA82Δ024 resulted in a marked and significant increase of up to 5.8-fold (P < 0.005) in luciferase activity relative to mock-infected cells (Fig. (Fig.3A).3A). These results indicate that expression of ORFV024 inhibits NF-κB-mediated gene transcription in ORFV-infected cells.

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 ...

ORFV024 suppresses NF-κB-regulated gene transcription following treatment of cells with LPS and TNF-α.

The ability of ORFV024 to inhibit NF-κB transcriptional activity was further investigated in primary OFTu and in HeLa cells. OFTu cells were cotransfected with a vector encoding for ORFV024-Flag fusion protein and with a plasmid encoding for a firefly luciferase reporter gene. Expression of ORFV024-Flag in LPS-treated OFTu cells significantly decreased NF-κB-regulated luciferase activity (~2-fold; P < 0.01) compared to control cells (Fig. 3B and C). Additionally, HeLa cells stably expressing GFP or ORFV024-GFP were transfected with the vector encoding the firefly luciferase reporter gene and treated with TNF-α for 6 h. NF-κB-mediated transcription of the luciferase gene was decreased approximately 4- to 5-fold (P < 0.05) in ORFV024-GFP-expressing cells compared to GFP-expressing controls (Fig. 3D and E). Together, these results indicate that ORFV024 inhibits activation of the NF-κB signaling pathway by two potent inducers, LPS and TNF-α.

ORFV024 decreases phosphorylation and nuclear translocation of NF-κB-p65 induced by TNF-α.

Given that posttranslational modifications define transcriptional functions of NF-κB-p65 in the nucleus (4, 36, 54), the effect of ORFV024 expression on NF-κB-p65 phosphorylation was investigated. HeLa cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa) and OFTu cells transfected with pCMVTag4A (empty) or pCMV024-Flag were treated with TNF-α and harvested at various times posttreatment. The expression of ORFV024 markedly decreased phosphorylation of NF-κB-p65 at Ser536 induced by TNF-α in both HeLa and OFTu cells (Fig. 4A and C). Densitometric analysis of blots probed with phosphor-NF-κB-p65 (Ser536) antibody revealed reductions of 51 and 66% in the phosphorylation of NF-κB-p65 in HeLa and OFTu cells, respectively (Fig. (Fig.4B,4B, P < 0.005; and Fig. Fig.4D,4D, P < 0.05). The reduced levels of phosphor-NF-κB-p65 are not due to protein degradation since levels of pan-NF-κB-p65 and Gβ were constant among all samples examined (Fig. 4A and C).

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, ...

In addition, nuclear translocation of NF-κB-p65 in response to TNF-α treatment was assessed in cells stably expressing GFP (GFP/HeLa) or ORFV024-GFP (024GFP/HeLa). Expression of ORFV024-GFP markedly decreased nuclear translocation of NF-κB-p65 in response to TNF-α-treatment compared to control GFP-expressing cells (Fig. (Fig.4E).4E). The decreased levels of NF-κB-p65 in the nucleus of 024GFP/HeLa cells were not due to protein degradation or to differences in protein loading, since levels of NF-κB-p65 in cell cytoplasm extracts were constant and no differences in the levels of the nuclear protein histone H3 were detected among the GFP and ORFV024-GFP samples. Together, the decreased levels of phosphor and nuclear translocated NF-κB-p65 demonstrate an inhibitory effect for ORFV024 on the NF-κB signaling pathway and further indicate the protein interferes with the pathway upstream of NF-κB-p65 phosphorylation.

ORFV024 decreases phosphorylation of IKKα and IKKβ in TNF-α-treated cells.

The crucial step on the activation of NF-κB signaling pathway is the phosphorylation and subsequent degradation of IκBα, which leads to release, posttranslational modification, and nuclear translocation of NF-κB-p65 (33, 53). To investigate whether the decreased levels of phosphor and nuclear translocated NF-κB-p65 detected in ORFV024-expressing cells were due to an interference with IκBα phosphorylation and/or degradation, GFP/HeLa and 024GFP/HeLa cells were examined for pan and phosphor levels of IκBα. ORFV024 expression was accompanied by a decrease in TNF-α-induced IκBα phosphorylation and, consequently, its degradation (Fig. (Fig.5A).5A). Densitometric analysis of blots probed with antibody against phosphor IκBα demonstrated a reduction of approximately 50% in IκBα phosphorylation in 024GFP/HeLa cells (Fig. (Fig.5B,5B, P < 0.005).

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 ...

IκB phosphorylation is mediated by upstream IκB kinases (33, 53). Given the importance of IKKs in triggering NF-κB activation and the decreased levels of phosphor IκBα detected in ORFV024-expressing cells, the potential role of ORFV024 as an inhibitor of IKK activation was investigated. ORFV024 expression markedly reduced phosphorylation of IKKα and IKKβ in 024GFP/HeLa cells (Fig. (Fig.6).6). These results indicate a role for ORFV024 in inhibiting IκBα phosphorylation and degradation by decreasing phosphorylation of IKK subunits IKKα/β.

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, ...

ORFV024 is nonessential for ORFV virulence in the natural host.

To investigate whether ORFV024 affects ORFV virulence, 4 to 5 months old, cross-breed lambs were inoculated with OV-IA82, OV-IA82Δ024, or MEM (control group). After local scarification, lambs were inoculated topically at the mucocutaneous junction of the lower lip and at the axillary skin and examined for the duration of the 19-day experiment. Characteristic clinical orf was observed in all virus-inoculated animals. Lesions developed by day 3 p.i. and consisted of erythema and papules, which evolved into vesicles and pustules at later times p.i. (Fig. (Fig.7).7). Local tissue proliferation and scab formation were first observed by days 5 and 7 p.i., respectively. Lesions started to subside on day 14 p.i. and by day 19 p.i. only limited scabs remained at lesion margins. No significant differences were observed in lesion severity, progression, or time to resolution between lambs inoculated with OV-IA82 or OV-IA82Δ024. Lambs from the control group did not exhibit changes other than mild scab formation due to skin scarification, which was completely resolved by day 4 p.i. (Fig. (Fig.77).

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 ...

Histopathological changes in axillary skin from virus-inoculated lambs consisted of marked epidermal hyperplasia, ballooning degeneration of keratinocytes in the stratum spinosum, hyperkeratosis, dyskeratosis, dermal leukocyte infiltration, occasional intraepithelial microabscesses, and accumulation of scale-crust (data not shown). No significant differences in the severity or time course of histological changes were observed between lambs inoculated with OV-IA82 or OV-IA82Δ024. Samples from mock-inoculated lambs were indistinguishable from normal skin. These results indicate that ORFV024 does not significantly affect ORFV virulence in the natural host.


Microarray and real-time PCR analysis of cells infected with deletion mutant virus OV-IA82Δ024 indicated that ORFV024 suppresses expression of NF-κB regulated proinflammatory genes, suggesting that ORFV024 inhibits activation of the NF-κB signaling pathway. Consistent with this hypothesis, we found a significant increase in expression of a NF-κB-responsive reporter gene in cells infected with OV-IA82Δ024 relative to those infected with wild-type virus. ORFV024 expression was shown here to suppress NF-κB-mediated transcription following treatment of cells with LPS or TNF-α. ORFV024 expression decreased TNF-α-induced phosphorylation and nuclear translocation of NF-κB-p65, phosphorylation and degradation of IκBα, and phosphorylation of IKK complex subunits IKKα and IKKβ. Together, these results indicate that ORFV024 prevents critical IKK activation, thus providing a mechanism for inhibition of NF-κB-mediated proinflammatory gene expression in infected cells.

Many poxviral proteins have been shown to inhibit activation of the IκB kinases, the bottleneck for most NF-κB-activating stimuli (11, 15, 24, 30, 45, 50, 62). For example, VACV proteins A46R, A52R, B14, and M2L interfere with activation of the IKK complex by antagonizing TLR signaling pathway (A46R and A52R), inhibiting activation of the IKK complex by the MEK/ERK signaling pathway (M2L), or directly binding to the IKK complex (B14L) (3, 11, 15, 24, 30, 62). In addition, VACV N1L presumably inhibits activation of the IKK complex by functioning upstream of IKKs, either at the level of adaptor protein TRAF6, or on downstream proteins that precede the IKK complex in the signaling cascade (26).

ORFV024 was shown here to inhibit activation of the IKK complex by decreasing phosphorylation of IKKα and IKKβ. Interaction of ORFV024 with IKKα, IKKβ, and IKKγ was investigated as a potential mechanism to prevent IκB kinase phosphorylation. However, no association between ORFV024 and IKKα, IKKβ, or IKKγ was detected (data not shown), suggesting that ORFV024 targets steps upstream of the IKK complex. Given that ORFV024 decreased expression of a NF-κB-responsive reporter gene following stimulation by both LPS and TNF-α, it is likely that this protein has evolved to interfere with activation of both, TLRs and TNFR signaling pathways (34, 65). The lack of homology between ORFV024 and other poxviral NF-κB inhibitors or any other molecule involved in the NF-κB signaling pathway, precludes assumptions on the precise mechanism by which ORFV024 interferes with IKK activation.

Like many poxviral genes encoding immune evasion and immunomodulatory proteins (1), including NF-κB inhibitors (42), transcription of ORFV024 in cultured cells was detected throughout ORFV infection cycle, indicating that it belongs to the early class of viral genes (1). ORFV024-GFP fusion protein exhibited a punctate distribution in the cytoplasm of stably expressing cells, and no colocalization of ORFV024-GFP with markers for the nucleus and specific cellular organelles was observed. This is likely of biological significance given that NF-κB activation pathways affected by ORFV024 take place in the cell cytoplasm (reviewed by 33).

Replication characteristics of OV-IA82Δ024 in cultures of OFTu and HeLa cells were similar to those of OV-IA82, indicating that ORFV024 is not essential for ORFV replication in these cells, and that like other poxviral NF-κB inhibitors (2, 11, 30, 41, 62), ORFV024 is dispensable for virus replication in vitro. Notably, OV-IA82Δ024 exhibited a distinct CPE (cell rounding and arrangement of cells in grapelike clusters) and plaque morphology (large cell-free central area and absence of flattened cells) in OFTu cells. These differences are likely explained by the increased expression of NF-κB-regulated genes induced by OV-IA82Δ024 infection, since activation of the NF-κB signaling pathway, and consequent gene expression, have been implicated in morphological and cytoskeletal changes in microglial cells after exposure to dsRNA (47) and in human endothelial cells expressing a viral activator of the NF-κB pathway (K13 protein from human herpesvirus 8) (37). The mechanisms by which ORFV024 influences cell morphology and cytoskeleton organization remain to be determined.

Given that the NF-κB signaling pathway integrates many aspects of the host innate and adaptive immunity (56), it was surprising that deletion of ORFV024 from the OV-IA82 genome had no significant effect on disease severity, progression, and resolution in sheep, indicating that ORFV024 is nonessential for virus virulence in the natural host. Although selected poxviral NF-κB inhibitors have been shown to affect virus virulence and pathogenesis to some extent (2, 11, 30, 41, 62), the degree of attenuation after single viral gene deletions has been modest and variable. For example, VACV B14R was shown to contribute to virus virulence in an intranasal but not intradermal murine model of infection (10), while deletion of VACV A46R, A52R, or N1L, rendered the virus partially attenuated in a mouse intranasal model (2, 30). In contrast, deletion of ankyrin repeat-encoding myxoma virus M150R and cowpox virus CPXV006 genes resulted in marked virus attenuation in rabbit intradermal and mouse intratracheal models of infection, respectively (6, 42). These observations indicate that the contribution of poxviral NF-κB inhibitors to virus infection and disease pathogenesis is complex and further suggest that multiple inhibitors encoded by individual poxviruses may exert complementary, perhaps partially overlapping functions to effectively evade NF-κB-mediated host immune responses.

The results presented here, showing that ORFV024 deletion had no significant effect on ORFV pathogenesis in sheep, further support the possibility for complementary functions by poxviral NF-κB inhibitors during infections in vivo. Indeed, we have identified two additional ORFV-encoded genes that interfere with the NF-κB signaling pathway (unpublished data), suggesting that ORFV NF-κB inhibitors may function in a coordinated and complementary fashion on distinct branches of the NF-κB signaling cascade to effectively suppress NF-κB-mediated host immune responses in vivo. Given the potential complementary actions of multiple poxviral NF-κB inhibitors, single viral gene deletions may not result in significant effects on virus virulence and pathogenesis.

Alternatively, ORFV024 may function in other, less-understood aspects of host-ORFV interactions. For example, persistent and/or subclinical infections have been described for ORFV and BPSV (32, 49, 58). The high prevalence of antibodies against parapoxviruses in herds and flocks with no history of clinical infections (35; unpublished data) and the ability of ORFV to persist in a flock in the absence of clinical manifestation of infection (49) suggest that persistent or subclinical infections likely play a role in the maintenance of parapoxviruses in nature. It is tempting to speculate that ORFV NF-κB inhibitors such as ORFV024 may contribute to aspects of virus persistence and transmission in the absence of overt viral infection.

The present study demonstrates that the novel ORFV protein ORFV024 interferes with activation of the NF-κB signaling pathway, while not significantly affecting virus virulence in the natural host. This represents the first description of an NF-κB inhibitor for ORFV or any other member of the Parapoxvirus genus. An improved understanding of ORFV functions associated with modulation and manipulation of host immune responses may contribute to increase current knowledge about the molecular aspects of ORFV biology and host-virus interactions.


We thank T. Shors (Department of Biology and Microbiology, University of Wisconsin-Oshkosh), M. Varela and P. R. Murcia (Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, United Kingdom), D. Refojo (Max Planck Institute of Psychiatry, Munich, Germany), and J. Shisler (Department of Microbiology, University of Illinois) for providing plasmids used in the present study.


[down-pointing small open triangle]Published ahead of print on 10 February 2010.


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