Poxvirus Vascular Endothelial Growth Factor (VEGF) Homologs of Orf Virus Grant McFadden1,* and Richard Moyer2 1
The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519)663-3184, fax: (519)663-3847, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.03015.
SUMMARY To date, a viral homolog of vascular endothelial growth factor (VEGF) has been discovered in only one poxvirus, namely orf virus of sheep. Orf virus encodes two such proteins, each of which possesses characteristic cysteine spacing motifs found in cellular VEGF family members. Viral VEGF binds to the members of the same family of cell surface receptors as its cellular counterparts and has proangiogenic activity.
BACKGROUND
Discovery Orf virus (OV) is a member of the parapoxvirus genus, a group of poxviruses which primarily infect ungulates and livestock (Robinson and Balassu, 1981). The virus causes contagious pustular dermatitis in sheep and goats and can be transmitted to humans (Mercer et al., 1997). The 139 kb OV genome, typical of parapoxviruses, is somewhat smaller and more G+C rich (63%) than poxviruses of the more widely studied and
distinct orthopoxvirus genus (36% G+C) (vaccinia virus Copenhagen, 192 kb, smallpox variola virus (Bangladesh), 186 kb) and exhibits a rather narrow host range (Gassmann et al., 1985; Robinson et al., 1987). Two distinct OV VEGF genes are known. They were discovered initially by routine sequencing derived from New Zealand isolates, strains NZ2 and NZ7 respectively (Lyttle et al., 1994). The NZ2 gene encodes a 133 amino acid (14.7 kDa) polypeptide; the NZ7 gene encodes a 148 amino acid (15 kDa) polypeptide.
Structure NZ2 and NZ7 are contiguous genes lacking introns.
Main activities and pathophysiological roles The role of the two viral genes within the context of the individual viruses is believed to be similar and responsible for the marked increase in capillary endothelial proliferation seen in the dermis following infection (Balassu and Robinson, 1987).
806 Grant McFadden and Richard Moyer
GENE AND GENE REGULATION
Accession numbers NZ2 VEGF: S67520 NZ7 VEGF: S67522
Genepept: NZ2: 1718158 NZ7: 1718159
Sequence See Figure 1.
Chromosome location The NZ7 and NZ2 VEGF genes are colocated in a similar region of the viral chromosome in both viruses. Unlike the gene designation for the orthopoxviruses, which is based on the HindIII restriction map, the OV virus map is based in a BamHI restriction map (Fleming et al., 1993). The largest viral DNA fragment resulting from BamHI digestion, the BamHI A fragment, is located at the right most region of the OV genome, and includes a portion of the inverted terminal repeat sequence found at both left and right extremes of the viral DNA. This region of the OV genome is the most divergent between the NZ2 and NZ7 strains. The A1R, A2R, and A3R open reading frames are the first three ORFs, reading from right to left, in the BamHI A fragment. The designation `R' refers to the direction of transcription, which is from left to right. The VEGF genes comprise ORF A2. Orf A3R is related to the 30 end of the vaccinia gene F9L.
Description of protein
Cells and tissues that express the gene
The two OV VEGF genes are derived from two distinct OV viruses. The two proteins are 73% similar and 41% identical to each other. The NZ7 gene is 148 amino acids, the NZ2 gene 133 amino acids. No introns are found in poxvirus genes. Both proteins have homology to the VEGF family and the B chain of the related platelet-derived growth factor (PDGF) family and the v-sis gene of simian sarcoma virus. Both OV proteins also share a sequence of N-terminal hydrophobic residues preceded by a lysine at residue 2, a putative cleavage site at residues 20Ð22 (Ala-Asp-Ser) in the NZ2 protein and the sequence (Ser-Gln-Ser) at residues 25Ð27 in the NZ7 protein. The remaining sequence of both proteins contains a region homologous to the v-sis minimal transforming region, which is conserved in all members of the VEGF family. This minimal transforming sequence of the two OV VEGF proteins retains the eight cysteine residues conserved in VEGF, two of which are needed for dimerization and functionality (amino acids 114Ð114 NZ2 VEGF, 130Ð132 NZ7 VEGF). A single N-linked glycosylation site is present (amino acids 85Ð87 NZ2 VEGF, 95Ð97 NZ7 VEGF). The glycosylation sites align with those in cellular VEGFs. Missing in both proteins are four blocks of sequence corresponding to the proposed central four exons in the human VEGF gene, including the highly basic sequence of 24 residues shown to be responsible for the cellassociated forms of human VEGF.
Poxvirus gene expression is independent of the infected cell.
Important homologies
Regulatory sites and corresponding transcription factors Expression of the VEGF gene is `early', before viral DNA replication.
See Table 1.
PROTEIN
Accession numbers GenBank: NZ2: S67520 NZ7: S67522
Posttranslational modifications Potential N-terminal cleavage at amino acids 22Ð22 in NZ2 VEGF, 25Ð27 in NZ7 VEGF; N-linked glycosylation site at NZ2 VEGF amino acids 85Ð87, NZ7 amino acids 95Ð97.
Poxvirus Vascular Endothelial Growth Factor (VEGF) Homologs of Orf Virus
807
Figure 1 Alignment of NZ7, NZ2, and v-sis from simian sarcoma virus.
Table 1 Comparative homologies of NZ2 and NZ7 VEGF proteins to various mammalian VEGFs showing similarities and identities NZ2 VEGF
NZ7 VEGF
Human VEGF
Rat VEGF
Guinea pig VEGF
Human placental VEGF
Similarities
Ð
73.3
56.6
50.3
43.8
55.3
Identities
Ð
41.1
28.5
24.6
24.6
27.5
Similarities
73.3
Ð
60.1
51.7
43.7
57.9
Identities
41.1
Ð
23.0
19.0
16.1
19.6
NZ2 VEGF
NZ7 VEGF
Extracted from data of Lyttle et al. (1994).
RECEPTOR UTILIZATION The NZ7 VEGF ligand is expressed as a 20 kDa dimer that binds to VEGF receptor 2 (KDR/Flk-1)
and induces receptor autophosphorylation, but does not bind to the VEGF receptor 1 (Flt-1) (Ogawa et al., 1998). The viral ligand cannot bind heparin, but exhibits comparable ability to stimulate primary
808 Grant McFadden and Richard Moyer endothelial cells into mitosis and induces vascular permeability as well as the cellular VEGF.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles It is believed that the protein functions to induce the marked capillary endothelial cell proliferation seen in the dermis, as well as the dilatation and dermal swelling seen in histological sections of lesions following OV infection.
References Balassu, T. C., and Robinson, A. J. (1987). Orf virus replication in bovine testis cells: kinetics of viral DNA, polypeptide, and
infectious virus production and analysis of virion polypeptides. Arch. Virol. 97, 267Ð281. Fleming, S. B., Blok, J., Fraser, K. M., Mercer, A. A., and Robinson, A. J. (1993). Conservation of gene structure and arrangement between vaccinia and orf virus. Virology 195, 175Ð184. Gassmann, U., Wyler, R., and Wittek, R. (1985). Analysis of parpoxvirus genomes. Arch. Virol. 83, 17Ð31. Lyttle, D. J., Fraser, K. M., Fleming, S. B., Mercer, A. A., and Robinson, A. J. (1994). Homologs of vascular endothelial growth factor are encoded by the poxvirus ORF virus. J. Virol. 68, 84Ð92. Mercer, A., Fleming, S., Robinson, A., Nettleton, P., and Reid, H. (1997). Molecular genetic analyses of parpoxviruses pathogenic for humans. Arch. Virol. 13, 25Ð34. Ogawa, S., Oku, A., Sawano, A., Yamaguchi, S., Yazaki, Y., and Shibuya, M. (1998). A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/ FLK-1 receptor and carries a potent mitotic activity without heparin-binding domain. J. Biol. Chem. 273, 31273Ð31282. Robinson, A. J., and Balassu, T. C. (1981). Contagious pustular dermatitis (orf). Vet. Bull. 51, 771Ð779. Robinson, A. J., Barns, G., Fraser, K., Carpenter, E., and Mercer, A. A. (1987). Conservation and variation in orf virus genomes. Virology 157, 13Ð23.
