Stephen B. Fleming

Viral Vascular Endothelial Growth Factor Plays a Critical Role in Orf Virus Infection

ABSTRACT Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.

Orf Virus Encodes a Novel Secreted Protein Inhibitor of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2

ABSTRACT The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a Kd of 369 pM and ovine IL-2 with a Kd of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

F-box-like domains are present in most poxvirus ankyrin repeat proteins.

Vertebrate poxviruses encode numerous proteins with the ankyrin (ANK) repeat, protein–protein interaction motif but little is known about the role(s) of this large family of poxvirus proteins. We report here that the vast majority of poxvirus ANK repeat proteins share a general molecular architecture that includes a conserved amino acid motif at the carboxyl terminus. This motif is most like the F-box seen in a range of cellular proteins. From 80–100% of the ANK repeat proteins of any one poxvirus have an F-box-like domain and we observed only one poxvirus protein with an F-box-like domain but lacking ANK repeats. The proteins of only one genus of vertebrate poxviruses lack F-box-like domains and this genus does not encode ANK repeat proteins. Many F-box proteins are recognition subunits of ubiquitin ligase complexes in which the F-box binds to core elements of the complex and protein–protein interaction domains in the remainder of the protein bind the substrate protein. These observations suggest a general model of the function of the poxvirus ANK-F-box proteins. We propose that the F-box-like domains in these proteins interact with cellular ubiquitin ligase complexes and thereby direct the ubiquitination of proteins bound to the ANK repeats. The large number of different poxviral ANK-F-box proteins suggests a wide range of cellular proteins might be subjected to ubiquitin-mediated degradation, thereby modulating diverse cellular responses to viral infection.

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cells ubiquitin-proteasome machinery.

Viral Vascular Endothelial Growth Factors Vary Extensively in Amino Acid Sequence, Receptor-binding Specificities, and the Ability to Induce Vascular Permeability yet Are Uniformly Active Mitogens

Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.

Analysis of an orf virus chemokine-binding protein: Shifting ligand specificities among a family of poxvirus viroceptors

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 granulocyte–macrophage colony-stimulating factor/IL-2 inhibitory factor also produced by ORFV, implicating the granulocyte–macrophage colony-stimulating factor/IL-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.

Sequence and Functional Analysis of a Homolog of Interleukin-10 Encoded by the Parapoxvirus Orf Virus

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 IL-10s and in this respect resemble 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 Th1 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-γ 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.

A Novel Bcl-2-Like Inhibitor of Apoptosis Is Encoded by the Parapoxvirus Orf Virus

ABSTRACT Apoptotic cell death forms part of the host defense against virus infection. We tested orf virus, a member of the poxvirus family, for the ability to inhibit apoptosis and found that orf virus-infected cells were fully resistant to UV-induced changes in cell morphology, caspase activation, and DNA fragmentation. By using a library of vaccinia virus-orf virus recombinants, we identified an orf virus gene (ORFV125) whose presence was linked with the inhibition of apoptosis. The 173-amino-acid predicted protein had no clear homologs in public databases other than those encoded by other parapoxviruses. However, ORFV125 possessed a distinctive C-terminal domain which was necessary and sufficient to direct the protein to the mitochondria. We determined that ORFV125 alone could fully inhibit UV-induced DNA fragmentation, caspase activation, and cytochrome c release and that its mitochondrial localization was required for its antiapoptotic function. In contrast, ORFV125 did not prevent UV-induced activation of c-Jun NH2-terminal kinase, an event occurring upstream of the mitochondria. These features are comparable to the antiapoptotic properties of the mitochondrial regulator Bcl-2. Furthermore, bioinformatic analyses revealed sequence and secondary-structure similarities to Bcl-2 family members, including characteristic residues of all four Bcl-2 homology domains. Consistent with this, the viral protein inhibited the UV-induced activation of the proapoptotic Bcl-2 family members Bax and Bak. ORFV125 is the first parapoxvirus apoptosis inhibitor to be identified, and we propose that it is a new antiapoptotic member of the Bcl-2 family.

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure

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 41.1% 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 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. 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.

Molecular genetic analyses of parapoxviruses pathogenic for humans

The current members of the genus parapoxvirus are orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV) and parapoxvirus of red deer in New Zealand (PVNZ). BPSV and PCPV are maintained in cattle while ORFV is maintained in sheep and goats, but all three are zoonoses. Only the recently reported PVNZ has yet to be recorded as infecting humans. Tentative members of the genus are camel contagious ecthyma virus, chamois contagious ecthyma virus and sealpoxvirus. The separation of the parapoxviruses into 4 distinct groups has been based on natural host range, pathology and, more recently, on restriction endonuclease and DNA/DNA hybridisation analyses. The latter studies have shown that the parapoxviruses share extensive homology between central regions of their genomes, but much lower levels of relatedness within the genome termini. The high G + C content of parapoxvirus DNA is in contrast to most other poxviruses and suggests that a significant genetic divergence from other genera of this family has occurred. DNA sequencing of portions of the genome of ORFV, the type species of the genus, has allowed a detailed comparison with the fully sequenced genome of the orthopoxvirus, vaccinia virus (VACV). These studies have provided a genetic map of ORFV and revealed a central core of 88 kbp within which the genomic content was strikingly similar to that of VACV. This conservation is not maintained in the genome termini where insertions, deletions and translocations have occurred. The characterisation of specific ORFV genes may lead to the construction of attenuated vaccine strains in which genes such as those with the potential to interfere with the immune response of the host have been deleted. The current ORFV vaccines are living unattenuated virus and vaccination lesions produce virus which contaminates the environment in a manner similar to natural infection. The virus in scab material is relatively resistant to inactivation and this virus both perpetuates the disease in sheep and provides the most likely source of human infections. A vaccine which immunises animals without perpetuating the disease could be the best way of reducing the incidence of ORFV infection of humans. It is likely that protection against infection by ORFV is cell mediated and will require the endogenous production of relevant antigens. We have recently constructed a series of VACV recombinants each of which contains a large multigene fragment of ORFV DNA. Together the recombinants represent essentially all of the ORFV genome in an overlapping manner. Vaccination of sheep with the recombinant library provided protection against challenge with virulent ORFV. Further studies with this library may enable dominant protective antigens of ORFV to be identified and lead to their incorporation into a subunit vaccine.