The parapoxvirus Orf virus inhibits dsDNA-mediated type I IFN expression via STING-dependent and STING-independent signalling pathways.

Type I interferons (IFNs) are critical in the host defence against viruses. They induce hundreds of interferon-stimulated genes (ISGs) many of which have an antiviral role. Poxviruses induce IFNs via their pathogen-associated molecular patterns, in particular, their genomic DNA. In a majority of cell types, dsDNA is detected by a range of cytoplasmic DNA sensors that mediate type I IFN expression via stimulator of interferon genes (STING). Orf virus (ORFV) induces cutaneous pustular skin lesions and is the type species of the Parapoxvirus genus within the Poxviridae family. The aim of this study was to investigate whether ORFV modulates dsDNA-induced type I IFN expression via STING-dependent signalling pathways in human dermal fibroblasts (hNDF) and THP-1 cells. We showed that ORFV infection of these cell types treated with poly(dA:dT) resulted in strong inhibition of expression of IFN-ß. In hNDFs, we showed using siRNA knock-down that STING was essential for type I IFN induction. IFN-ß expression was further reduced when both STING and RIG-I were knocked down. In addition, HEK293 cells that do not express STING or Toll-like receptors also produce IFN-ß following stimulation with poly(dA:dT). The 5' triphosphate dsRNA produced by RNA polymerase III specifically results in the induction of type I IFNs through the RIG-I receptor. We showed that ORFV infection resulted in strong inhibition of IFN-ß expression in HEK293 cells stimulated with poly(dA:dT). Overall, this study shows that ORFV potently counteracts the STING-dependent and STING-independent IFN response by antagonizing dsDNA-activated IFN signalling pathways.

The parapoxvirus Orf virus inhibits IFN-ß expression induced by dsRNA.

Orf virus (ORFV) is the type species of the Parapoxvirus genus that belongs to the Poxviridae family. Type I interferons (IFN) are critical in the host defence against viruses. They induce hundreds of interferon stimulated genes (ISGs) many of which have an antiviral role. The ability of ORFV to modulate type I IFN production was undertaken to investigate whether ORFV could inhibit IFN-ß expression via dsRNA dependant signalling pathways. HEK293 cells are known to lack DNA pattern-recognition receptors and Toll-like receptors however, they do express the cytosolic dsRNA receptors RIG-I and MDA5. HEK293 cells were shown to produce high levels of IFN-ß when cells were stimulated with poly(I:C) and this was shown to be predominantly via RIG-I-dependant signalling as confirmed by siRNA knock-down of RIG-I. Further we showed that HEK293 cells are permissive for ORFV and caused potent inhibition of IFN-ß transcription when cells were stimulated with poly(I:C) post-viral infection. Studies using heat inactivated ORFV suggested that de novo synthesis of early genes was required. In addition our findings showed that the ORFV encoded factor ORF020, that is known to bind dsRNA, is involved in antagonising IFN expression. Overall, this study has shown for first time the ability of ORFV to counteract type I IFN expression by antagonising dsRNA-activated RIG-I signalling.

The parapoxvirus Orf virus ORF116 gene encodes an antagonist of the interferon response.

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the Poxviridae family. Genetic and functional studies have revealed ORFV has multiple immunomodulatory genes that manipulate innate immune responses, during the early stage of infection. ORF116 is a novel gene of ORFV with hitherto unknown function. Characterization of an ORF116 deletion mutant showed that it replicated in primary lamb testis cells with reduced levels compared to the wild-type and produced a smaller plaque phenotype. ORF116 was shown to be expressed prior to DNA replication. The potential function of ORF116 was investigated by gene-expression microarray analysis in HeLa cells infected with wild-type ORFV or the ORF116 deletion mutant. The analysis of differential cellular gene expression revealed a number of interferon-stimulated genes (ISGs) differentially expressed at either 4 or 6 h post infection. IFI44 showed the greatest differential expression (4.17-fold) between wild-type and knockout virus. Other ISGs that were upregulated in the knockout included RIG-I, IFIT2, MDA5, OAS1, OASL, DDX60, ISG20 and IFIT1 and in addition the inflammatory cytokine IL-8. These findings were validated by infecting HeLa cells with an ORF116 revertant recombinant virus and analysis of transcript expression by quantitative real time-PCR (qRT-PCR). These observations suggested a role for the ORFV gene ORF116 in modulating the IFN response and inflammatory cytokines. This study represents the first functional analysis of ORF116.

Rescue of a Vaccinia Virus Mutant Lacking IFN Resistance Genes K1L and C7L by the Parapoxvirus Orf Virus.

Type 1 interferons induce the upregulation of hundreds of interferon-stimulated genes (ISGs) that combat viral replication. The parapoxvirus orf virus (ORFV) induces acute pustular skin lesions in sheep and goats and can reinfect its host, however, little is known of its ability to resist IFN. Vaccinia virus (VACV) encodes a number of factors that modulate the IFN response including the host-range genes C7L and K1L. A recombinant VACV-Western Reserve (WR) strain in which the K1L and C7L genes have been deleted does not replicate in cells treated with IFN-ß nor in HeLa cells in which the IFN response is constitutive and is inhibited at the level of intermediate gene expression. Furthermore C7L is conserved in almost all poxviruses. We provide evidence that shows that although ORFV is more sensitive to IFN-ß compared with VACV, and lacks homologues of KIL and C7L, it nevertheless has the ability to rescue a VACV KIL- C7L- gfp+ mutant in which gfp is expressed from a late promoter. Co-infection of HeLa cells with the mutant and ORFV demonstrated that ORFV was able to overcome the block in translation of intermediate transcripts in the mutant virus, allowing it to progress to late gene expression and new viral particles. Our findings strongly suggest that ORFV encodes a factor(s) that, although different in structure to C7L or KIL, targets an anti-viral cellular mechanism that is a highly potent at killing poxviruses.

Orf Virus IL-10 and VEGF-E Act Synergistically to Enhance Healing of Cutaneous Wounds in Mice.

Orf virus (OV) is a zoonotic parapoxvirus that causes highly proliferative skin lesions which resolve with minimal inflammation and scarring. OV encodes two immunomodulators, vascular endothelial growth factor (VEGF)-E and interleukin-10 (ovIL-10), which individually modulate skin repair and inflammation. This study examined the effects of the VEGF-E and ovIL-10 combination on healing processes in a murine wound model. Treatments with viral proteins, individually and in combination, were compared to a mammalian VEGF-A and IL-10 combination. Wound biopsies were harvested to measure re-epithelialisation and scarring (histology), inflammation, fibrosis and angiogenesis (immunofluorescence), and gene expression (quantitative polymerase chain reaction). VEGF-E and ovIL-10 showed additive effects on wound closure and re-epithelialisation, and suppressed M1 macrophage and myofibroblast infiltration, while allowing M2 macrophage recruitment. The viral combination also increased endothelial cell density and pericyte coverage, and improved collagen deposition while reducing the scar area. The mammalian combination showed equivalent effects on wound closure, re-epithelialisation and fibrosis, but did not promote blood vessel stabilisation or collagen remodeling. The combination treatments also differentially altered the expression of transforming growth factor beta isoforms, Tgfß1 and Tgfß3. These findings show that the OV proteins synergistically enhance skin repair, and act in a complimentary fashion to improve scar quality.

Chemokine-Binding Proteins Encoded by Parapoxvirus of Red Deer of New Zealand Display Evidence of Gene Duplication and Divergence of Ligand Specificity.

Parapoxvirus of red deer in New Zealand (PVNZ) is a species of the Parapoxvirus genus that causes pustular dermatitis. We identified a cluster of genes in PVNZ that encode three unique chemokine-binding proteins (CBPs) namely ORF112.0, ORF112.3 and ORF112.6. Chemokines are a large family of molecules that direct cell trafficking to sites of inflammation and through lymphatic organs. The PVNZ-CBPs were analyzed by surface plasmon resonance against a broad spectrum of CXC, CC, XC and CX3C chemokines and were found to differ in their specificity and binding affinity. ORF112.0 interacted with chemokines from the CXC, CC and XC classes of chemokines with nM affinities. The ORF112.3 showed a preference for CXC chemokines, while ORF112.6 showed pM affinity binding for CC chemokines. Structural modeling analysis showed alterations in the chemokine binding sites of the CBPs, although the core structure containing two ß-sheets and three α-helices being conserved with the other parapoxvirus CBPs. Chemotaxis assays using neutrophils and monocytes revealed inhibitory impact of the CBPs on cell migration. Our results suggest that the PVNZ-CBPs are likely to have evolved through a process of gene duplication and divergence, and may have a role in suppressing inflammation and the anti-viral immune response.

VEGF Receptor-2 Activation Mediated by VEGF-E Limits Scar Tissue Formation Following Cutaneous Injury.

Objective: Vascular endothelial growth factor (VEGF) family members are critical regulators of tissue repair and depending on their distinct pattern of receptor specificity can also promote inflammation and scarring. This study utilized a receptor-selective VEGF to examine the role of VEGF receptor (VEGFR)-2 in scar tissue (ST) formation. Approach: Cutaneous skin wounds were created in mice using a 4 mm biopsy punch and then treated until closure with purified VEGF-E derived from orf virus stain NZ-2. Tissue samples were harvested to measure gene expression using quantitative PCR and to observe ST formation through histological examination and changes in cell populations by immunofluorescence. Results: VEGFR-2-activation with VEGF-E increased expression of anti-inflammatory cytokine interleukin (IL)-10 and reduced macrophage infiltration and myofibroblast differentiation in wounded skin compared with controls. VEGF-E treatment also increased microvascular density and improved pericyte coverage of blood vessels in the healing wounds. The ST that formed following treatment with VEGF-E was reduced in size and showed improved collagen structure. Innovation: The role of VEGFR-2 activation in wound epithelialization and angiogenesis is well established; but its contribution to ST formation is unclear. This study tests the effect of a selective VEGFR-2 activation on ST formation following cutaneous wounding in a murine model. Conclusion: VEGFR-2 stimulation can enhance the quality of skin repair, at least, in part, through the induction of IL-10 expression and dampening of wound inflammation and fibrosis. Therapies that selectively activate VEGFR-2 may therefore be beneficial to treat impaired healing or to prevent excess scarring.

Treatment of limb wounds of horses with orf virus IL-10 and VEGF-E accelerates resolution of exuberant granulation tissue, but does not prevent its development.

Bandaging of limb wounds in horses leads to formation of exuberant granulation tissue (EGT) that retards healing due to protracted inflammation, aberrant vascularisation and delayed epithelialisation. EGT is not observed if wounds are left undressed or when wounds are on the body. A previous study showed that short-term administration of proteins derived from orf virus dampened inflammation and promoted epithelialisation of open wounds in horses. Here, we investigated the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on the development and resolution of EGT. Excisional wounds were created on the forelimb of four horses, and bandages were maintained until full healing to induce EGT formation. Matching body wounds were created to ensure EGT was limited to the limb, and to differentiate the effects of the viral proteins on normal healing and on EGT formation. Viral proteins or the hydrogel vehicle control were administered topically to site-matched wounds at day 1, with repeat administration at day 8. Wound healing and EGT formation were monitored macroscopically. Wound margin samples were harvested at 2, 7 and 14 days, and at full healing, with histology used to observe epithelialisation, immunofluorescence used to detect inflammatory cells, angiogenesis and cell death, and qPCR to measure expression of genes regulating inflammation and angiogenesis. Limb wounds developed EGT, and exhibited slower healing than body wounds. Viral protein treatment did not accelerate healing at either location nor limit EGT formation in limb wounds. Treatment of limb wounds did however increase epithelialisation and angiogenesis, without dampening inflammatory cell infiltration or gene expression. The healed wounds also had less occlusion and death of blood vessels and fewer epidermal rete ridges following viral protein treatment. These findings indicate that the viral protein treatment does not suppress wound inflammation or EGT formation, but does promote vascular and epidermal repair and EGT resolution.

Deletion of the Chemokine Binding Protein Gene from the Parapoxvirus Orf Virus Reduces Virulence and Pathogenesis in Sheep.

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the family Poxviridae and infects sheep and goats, often around the mouth, resulting in acute pustular skin lesions. ORFV encodes several secreted immunomodulators including a broad-spectrum chemokine binding protein (CBP). Chemokines are a large family of secreted chemotactic proteins that activate and regulate inflammation induced leukocyte recruitment to sites of infection. In this study we investigated the role of CBP in vivo in the context of ORFV infection of sheep. The CBP gene was deleted from ORFV strain NZ7 and infections of sheep used to investigate the effect of CBP on pathogenesis. Animals were either infected with the wild type (wt) virus, CBP-knockout virus or revertant strains. Sheep were infected by scarification on the wool-less area of the hind legs at various doses of virus. The deletion of the CBP gene severely attenuated the virus, as only few papules formed when animals were infected with the CBP-knock-out virus at the highest dose (107 p.f.u). In contrast, large pustular lesions formed on almost all animals infected with the wt and revertant strains at 107 p.f.u. The lesions for the CBP-knock-out virus resolved approximately 5-6 days p.i, at a dose of 107 pfu whereas in animals infected with the wt and revertants at this dose, lesions began to resolve at approximately 10 days p.i. Few pustules developed at the lowest dose of 103 p.f.u. for all viruses. Immunohistochemistry of biopsy skin-tissue from pustules showed that the CBP-knockout virus replicated in all animals at the highest dose and was localized to the skin epithelium while haematoxylin and eosin staining showed histological features of the CBP-knockout virus typical of the parent virus with acanthosis, elongated rete ridges and orthokeratotic hyperkeratosis. MHC-II immunohistochemistry analysis for monocytes and dendritic cells showed greater staining within the papillary dermis of the CBP-knock-out virus compared with the revertant viruses, however this was not the case with the wt where staining was similar. Our results show that the CBP gene encodes a secreted immunodulator that has a critical role in virulence and pathogenesis.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

A Broad-Spectrum Chemokine-Binding Protein of Bovine Papular Stomatitis Virus Inhibits Neutrophil and Monocyte Infiltration in Inflammatory and Wound Models of Mouse Skin.

Bovine papular stomatitis virus (BPSV) is a Parapoxvirus that induces acute pustular skin lesions in cattle and is transmissible to humans. Previous studies have shown that BPSV encodes a distinctive chemokine-binding protein (CBP). Chemokines are critically involved in the trafficking of immune cells to sites of inflammation and infected tissue, suggesting that the CBP plays a role in immune evasion by preventing immune cells reaching sites of infection. We hypothesised that the BPSV-CBP binds a wide range of inflammatory chemokines particularly those involved in BPSV skin infection, and inhibits the recruitment of immune cells from the blood into inflamed skin. Molecular analysis of the purified protein revealed that the BPSV-CBP is a homodimeric polypeptide with a MW of 82.4 kDa whilst a comprehensive screen of inflammatory chemokines by surface plasmon resonance showed high-affinity binding to a range of chemokines within the CXC, CC and XC subfamilies. Structural analysis of BPSV-CBP, based on the crystal structure of orf virus CBP, provided a probable explanation for these chemokine specificities at a molecular level. Functional analysis of the BPSV-CBP using transwell migration assays demonstrated that it potently inhibited chemotaxis of murine neutrophils and monocytes in response to CXCL1, CXCL2 as well as CCL2, CCL3 and CCL5 chemokines. In order to examine the effects of CBP in vivo, we used murine skin models to determine its impact on inflammatory cell recruitment such as that observed during BPSV infection. Intradermal injection of BPSV-CBP blocked the influx of neutrophils and monocytes in murine skin in which inflammation was induced with lipopolysaccharide. Furthermore, intradermal injection of BPSV-CBP into injured skin, which more closely mimics BPSV lesions, delayed the influx of neutrophils and reduced the recruitment of MHC-II+ immune cells to the wound bed. Our findings suggest that the CBP could be important in pathogenesis of BPSV infections.

Short-term treatment of equine wounds with orf virus IL-10 and VEGF-E dampens inflammation and promotes repair processes without accelerating closure.

Healing is delayed in limb wounds relative to body wounds of horses, partly because of sustained inflammation and inefficient angiogenesis. In laboratory animals, proteins derived from orf virus modulate these processes and enhance healing. We aimed to compare immune cell trafficking and the inflammatory, vascular, and epidermal responses in body and limb wounds of horses and then to investigate the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on these processes. Standardized excisional wounds were created on the body and forelimb of horses and their progression monitored macroscopically until healed. Tissue samples were harvested to measure the expression of genes regulating inflammation and repair (quantitative polymerase chain reaction) and to observe epithelialization (histology), innate immune cell infiltration, and angiogenesis (immunofluorescence). Delayed healing of limb wounds was characterized by intensified and extended pro-inflammatory signaling and exacerbated innate immune response, concomitant with the absence of anti-inflammatory eIL-10. Blood vessels were initially more permeable and then matured belatedly, concomitant with retarded production of angiogenic factors. Epithelial coverage was achieved belatedly in limb wounds. Viral proteins were administered to wounds of one body and one limb site/horse at days 1-3, while wounds at matching sites served as controls. Treatment dampened pro-inflammatory gene expression and the innate immune response in all wounds. It also improved angiogenic gene expression, but primarily in body wounds, where it altered blood vessel density and myofibroblast persistence. Moreover, the viral proteins increased epithelialization of all wounds. The short-term viral protein therapy did not, however, improve the healing rate of wounds in either location, likely due to suboptimal dosing. In conclusion, we have further detailed the processes contributing to protracted healing in limb wounds of horses and shown that short-term administration of viral proteins exerts several promising though transient effects that, if optimized, may positively influence healing.

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

BACKGROUND: Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. HYPOTHESIS/OBJECTIVES: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. ANIMALS: Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. METHODS: Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. RESULTS: Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. CONCLUSIONS AND CLINICAL IMPORTANCE: This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses.

Orf virus IL-10 reduces monocyte, dendritic cell and mast cell recruitment to inflamed skin.

Orf virus (ORFV) is a zoonotic parapoxvirus that causes pustular dermatitis of sheep, and occasionally humans. Despite causing sustained infections, ORFV induces only a transient increase in pro-inflammatory signalling and the trafficking of innate immune cells within the skin seems to be impaired. An explanation for this tempered response to ORFV infection may lie in its expression of a homolog of the anti-inflammatory cytokine, interleukin (IL)-10. Using a murine model in which inflammation was induced by bacterial lipopolysaccharide, we examined the effects of the ORFV-IL-10 protein on immune cell trafficking to and from the skin. ORFV-IL-10 limited the recruitment of blood-derived Gr-1(int)/CD11b(int) monocytes, CD11c(+ve)/MHC-II(+ve) dendritic cells and c-kit(+ve)/FcεR1(+ve) mature mast cells into inflamed skin. ORFV-IL-10 also suppressed the activation of CD11c(+ve)/MHC-II(+ve) dendritic cells within the skin, reducing their trafficking to the draining lymph node. These findings suggest that expression of IL-10 by ORFV may contribute to the impaired trafficking of innate immune cells within infected skin.

Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway.

Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type I IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-γ or IFN-α respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-γ or IFN-α treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway.

Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a ß-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP ß-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP ß-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations.

Molecular genetic analysis of orf virus: a poxvirus that has adapted to skin.

Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 132 genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial growth factor genes have been "captured" from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating factor and interleukin-2 appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear factor (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in keratinocytes. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis.

Poxviral ankyrin proteins.

Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

Effect of a broad-specificity chemokine-binding protein on brain leukocyte infiltration and infarct development.

BACKGROUND AND PURPOSE: Expression of numerous chemokine-related genes is increased in the brain after ischemic stroke. Here, we tested whether post-stroke administration of a chemokine-binding protein (CBP), derived from the parapoxvirus bovine papular stomatitis virus, might reduce infiltration of leukocytes into the brain and consequently limit infarct development. METHODS: The binding spectrum of the CBP was evaluated in chemokine ELISAs, and binding affinity was determined using surface plasmon resonance. Focal stroke was induced in C57Bl/6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion for 23 or 47 hours. Mice were treated intravenously with either bovine serum albumin (10 µg) or CBP (10 µg) at the commencement of reperfusion. At 24 or 48 hours, we assessed plasma levels of the chemokines CCL2/MCP-1 and CXCL2/MIP-2, as well as neurological deficit, brain leukocyte infiltration, and infarct volume. RESULTS: The CBP interacted with a broad spectrum of CC, CXC, and XC chemokines and bound CCL2/MCP-1 and CXCL2/MIP-2 with high affinity (pM range). Stroke markedly increased plasma levels of CCL2/MCP-1 and CXCL2/MIP-2, as well as numbers of microglia and infiltrating leukocytes in the brain. Increases in plasma chemokines were blocked in mice treated with CBP, in which there was reduced neurological deficit, fewer brain-infiltrating leukocytes, and ≈50% smaller infarcts at 24 hours compared with bovine serum albumin-treated mice. However, CBP treatment was no longer protective at 48 hours. CONCLUSIONS: Post-stroke administration of CBP can reduce plasma chemokine levels in association with temporary atten uation of brain inflammation and infarct volume development.

Orf virus IL-10 accelerates wound healing while limiting inflammation and scarring.

Interleukin (IL)-10 plays a critical role in controlling wound inflammation and scar formation. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that resolve with minimal scarring. Orf virus encodes a range of factors that subvert the host's response to infection, including a homolog of IL-10. This study investigated, using a murine full-thickness wound model, whether purified orf virus IL-10 (ovIL-10) can regulate skin repair and scarring. Repeat injections of ovIL-10 into wounded skin accelerated wound closure. Histological analyses of wound sections revealed that treatment with ovIL-10 accelerated wound reepithelialization, granulation tissue coverage of the wound bed, and improved wound revascularization. In addition, wounds treated with ovIL-10 showed a reduction in macrophage infiltration, myofibroblast differentiation, and wound contraction. Treatment of wounds with ovIL-10 also resulted in a reduction in visible scarring that was consistent with the extent of scar tissue formed. Quantitative polymerase chain reaction analysis confirmed that ovIL-10 reduced the expression of key mediators of inflammation and granulation tissue formation. These findings show that ovIL-10, like mammalian IL-10, limits inflammation and scar tissue formation and reveal a new role for both mammalian and viral IL-10 in mediating tissue repair.