We identify a secreted chemokine inhibitor encoded by orf virus (ORFV), the prototypic poxvirus of the Parapoxvirus genus, and show that it is related to the poxvirus type II CC-chemokine-binding proteins (CBP-II) produced by members of the Orthopoxvirus and Leporipoxvirus genera. The ORFV chemokine-binding protein (CBP) is functionally similar to the CBP-II proteins in its ability to bind and inhibit many CC-chemokines with high affinity. However, unlike CBP-II, the ORFV CBP also binds with high affinity to lymphotactin, a member of the C-chemokine family, demonstrating that the ORFV CBP possesses an altered binding specificity. Interestingly, the amino acid sequence of ORFV CBP more closely resembles the granulocytemacrophage colony-stimulating factor͞IL-2 inhibitory factor also produced by ORFV, implicating the granulocyte-macrophage colonystimulating factor͞IL-2 inhibitory factor protein as a highly diverged, but related, member of the CBP-II protein family. Notably, these findings suggest that the genes that encode these proteins derive from a common poxvirus ancestral gene that has since been modified in binding specificity during speciation of the poxvirus genera. Overall, these findings illustrate the concept of evolution of viral proteins at the biophysical and molecular interface.
The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41n1 % amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30n8 % with an average amino acid identity between pairs of NZ2-like sequences of 86n1 %. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.
A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function.
Orf virus is a large DNA virus and is the type species of the Parapoxvirus genus of the family Poxviridae. Orf virus infects the epithelium of sheep and goats and is transmissible to humans. Recently we discovered a gene in orf virus that encodes a polypeptide with remarkable homology to mammalian interleukin (IL-10) and viral encoded IL-10s of herpes viruses. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%) and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpes virus (67%). The C-terminal region, comprising two-thirds of the orf virus protein, is identical to ovine IL-10 which suggests that this gene has been captured from its host sheep during the evolution of orf virus. In contrast the N-terminal region shows little homology with cellular IL10s and in this respect resembles other viral IL-10s. IL-10 is a pleiotrophic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. IL-10 is a potent anti-inflammatory cytokine with inhibitory effects on non-specific immunity in particular macrophage function and Thl effector function. Our studies so far, indicate, that the functional activities of orf virus IL-10 are the same as ovine IL-10. Orf virus IL-10 stimulates mouse thymocyte proliferation and inhibits cytokine synthesis in lipopolysaccharide-activated ovine macrophages, peripheral blood monocytes and keratinocytes. Infection of sheep with an IL-10 deletion mutant of orf virus has shown that interferon-gamma levels are higher in tissue infected with the mutant virus than the parent virus. The functional activities of IL-10 and our data on orf virus IL-10 suggest a role in immune evasion.
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.
Orf virus (ORFV) is the type species of the parapoxvirus genus and produces cutaneous pustular lesions in sheep, goats and humans. The genome encodes a polypeptide with remarkable homology to interleukin-10 (IL-10), particularly ovine IL-10, and also to IL-10-like proteins encoded by Epstein-Barr virus (EBV) and equine herpesvirus. IL-10 is a pleiotropic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. We have expressed and purified C-terminal FLAG and His 6 -tagged versions of ORFV-IL-10 and shown that ORFV-IL-10 costimulates murine mast cells (MC/9) and inhibits tumour necrosis factor-α synthesis in activated mouse peritoneal macrophages. Our results demonstrate that although ORFV-IL-10 is structurally similar to EBV-IL-10 it has evolved a different spectrum of activities. EBV-IL-10 does not stimulate the proliferation of thymocytes or mast cells whereas ORFV-IL-10 has both of these activities. Recent studies show that the critical difference in molecular structure of human IL-10 and EBV-IL-10, which may be the basis of their functional differences, is linked to a single amino acid substitution. Consistent with the activity spectrum reported here for ORFV-IL-10, the viral gene encodes the critical amino acid seen in human IL-10. Although the ORFV-IL-10 gene has clearly undergone significant evolutionary change at the nucleotide level compared with ovine IL-10, it has largely retained the polypeptide structure and functional characteristics of its ovine counterpart, suggesting that mutations of the gene to a potentially more potent immunosuppressive form may compromise the co-existence of host and virus.
Orf virus is the prototype parapoxvirus that causes the contagious skin disease orf. It encodes an orthologue of the cytokine interleukin (IL)-10. Recombinant orf viruses were constructed in which the viral interleukin-10 (vorfIL-10) was disabled (vorfIL-10ko) and reinserted (vorfrevIL-10) at the same locus and compared to wild-type virus for their ability to induce skin lesions in sheep. After either primary infection or reinfection, smaller less severe lesions were recorded in the vorfIL-10ko-infected animals compared with either of the vorfIL-10-intact virus-infected animals. Thus, the vorfIL-10ko virus was attenuated compared with the vorfIL-10 intact viruses, demonstrating that orf virus IL-10 is a virulence factor. The virus IL-10 is one of several virulence or immuno-modulatory factors expressed by orf virus. Removal of any one of these genes would be expected to have only a partial effect on virulence, which is what was observed in this study with vorfIL-10.
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