1 Federal Research Centre for Virus Diseases of Animals, Institute of Immunology, Paul-Ehrlich-Straße 28, D-72076 Tübingen, Federal Republic of Germany
2 Divison of Molecular Microbiology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Correspondence
Hanns-Joachim Rziha
achim.rziha{at}tue.bfav.de
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ABSTRACT |
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The DNA sequences of D1701 (9979 nucleotides) and of B177 (7702 nucleotides) are deposited in GenBank under accession nos AY186732 and AY186733, respectively.
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MAIN TEXT |
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Due to a rearrangement of terminal sequences, the highly attenuated ORFV strain D1701 contains two copies of the VEGF-E and IL-10 genes (Cottone et al., 1998; Rziha et al., 1999
). Recently, it was suggested that BPSV, PCPV and PVNZ do not harbour an ORFV-like IL-10 gene (Fleming et al., 2000
). Data on the presence of the VEGF-E gene in PPV other than ORFV do not exist. Various DNA hybridization experiments have indicated the absence of VEGF-E homologous sequences in the genome of the BPSV isolate B177. This virus isolate was obtained from a typical non-proliferative BPS lesion from a calf muzzle by inoculation of foetal bovine kidney cells. Before plaque purification and isolation of viral DNA, B177 was propagated a maximum of three times in the bovine kidney cell line BK-KL3A (Cottone et al., 1998
). Southern blot hybridization using 32P-labelled probes was performed as described by Cottone et al. (1998)
. The plasmid pVEGF was used as a probe specific for the D1701 VEGF-E gene (Meyer et al., 1999
), which shows 90 % nucleotide identity to that of ORFV strain NZ2 (Mercer et al., 2002
). The probe specific for the ORFV strain NZ7 was obtained from plasmid pSB161 (generously provided by S. Fleming, University of Otago, New Zealand). Using various stringencies of annealing conditions, neither the probe specific for the D1701-type nor the NZ7-type VEGF-E gene reacted with any DNA restriction fragment from BPSV B177, although under relaxed conditions the NZ7-type-specific probe detected the D1701 counterpart (data not shown). In contrast, genes adjacent to and upstream of D1701 VEGF-E (e.g. F9L or F10L; Fig. 1
) could be detected easily with B177 DNA (data not shown). Only very weak hybridization was found with probes covering the D1701 ANK-2 and ANK-3 genes (Fig. 1B
).
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The relationship of the ORF3 genes is assumed from the marked amino acids similarity between D1701 ORF3 and the 95 sequenced amino acids of B177 (Table 1). The early ORF3 gene was first described for ORFV NZ2 and NZ7 (Fleming et al., 1991
; Fraser et al., 1990
) and displays 76·2 % amino acid identity to that of D1701. This gene of unknown function does not show homology to any other poxviral gene and seems to be unique for PPV. Beyond the ORF3 gene, the available
1000 bases of the D1701 sequence show no homology to that of NZ2 or NZ7 (not shown). Upstream of the F10L gene, three so far unknown genes were found in ORFV D1701 and BPSV B177. Database searches using Pfam alignment showed that all three potential genes contained tandemly arranged stretches of 3344 amino acids in length characteristic of ankyrin repeats and have been tentatively designated as ANK-1 to ANK-3 (Fig. 1
). From the available sequence data, the C-terminal 211 amino acids of the B177 ANK-1 gene beginning with the last of eight ankyrin repeats seen in D1701 indicate considerable similarity to D1701 ANK-1 (Table 1
). The ANK-2 and ANK-3 genes are of similar size and contain eight or nine ankyrin repeat motifs, and the deduced amino acid sequences show 62·5 or 66·9 % amino acid similarity, respectively (Table 1
). The lower DNA identity (maximally 65 %) between D1701 and B177 of the ANK-2 and ANK-3 gene region explains missing or weak hybridization signals with specific probes. Although the DNA homology of the described genes varies between both viruses, the average G+C content of this BPSV genome seems high, comparable with ORFV (Wittek et al., 1979
). The existence of counterparts of the new ANK genes in other PPV is unknown. Apart from the presence of the ankyrin repeat motifs, the three ANK genes showed no similarity to the G1L gene, which is the first reported ankyrin-repeat-containing ORFV gene and maps at the left terminus of the genome (Cottone et al., 1998
; Sullivan et al., 1995b
). A number of ankyrin-repeat-containing proteins have been identified in various poxviruses, also located at near-terminal genomic regions, and are expressed early in infection. Early expression of the new ANK genes, which was expected from the presence of sequence motifs resembling poxviral early promoters and the canonical early transcription stop motif (Fig. 1
), was found for ANK-2 and ANK-3 in D1701-infected cells (data not shown). Numerous prokaryotic and eukaryotic proteins acting as protein linkers use the ankyrin repeats as binding domains for proteinprotein interactions, thereby influencing cell-cycle control, cell differentiation and host immunity (Ghosh et al., 1998
; Rubtsov & Lopina, 2000
). The cowpoxvirus-encoded ankyrin repeat protein gene CHOhr determines cell tropism (Spehner et al., 1988
) and functions as an anti-apoptotic gene (Ink et al., 1995
). Whether PPV ANK genes can also mediate proteinprotein interactions and exert important functions for the tropism or pathobiology of ORFV awaits further investigation. Additionally, it would be interesting to see whether other PPV also display a collinear genomic arrangement of related ANK genes, which might further indicate their importance for the PPV life cycle.
The recently described ORFV IL-10 gene was also detected in ORFV D1701 and BPSV B177 (Fig. 2 and Table 1
). Most sequence differences were found in the first 50 amino acids, whereas the final two-thirds of the proteins show the highly conserved IL-10 core motifs (Fig. 2
). Signal peptide cleavage sites, identified using program SignalP, could be located in the variable N terminus of the proteins. Remarkably, the IL-10 genes exhibited a noticeably low G+C content compared with the flanking sequence (Table 1
), supporting earlier suggestions that those genes acquired from the natural host have retained the low G+C content (Fleming et al., 1997
, 2000
; Lyttle et al., 1994
). This was supported by the comparison of amino acid relationships. Homology searches using the bic2 program (EMBL) revealed a higher score of BPSV IL-10 to bovine IL-10 (score 703) over a range of 139 amino acids (aa 52189) than to ovine IL-10 (score 678). Closer inspection of the amino acid sequences showed that B177 and bovine IL-10 possess identical amino acids at six positions within the conserved part of the protein (Fig. 2
, asterisks). At these positions the three ORFV strains exhibited residues that differ from the bovine IL-10 but are identical to the ovine IL-10, explaining the closer relationship of ORFV and ovine IL-10. One of the amino acids exclusively shared by the bovine and BPSV IL-10 is predicted to interact with the IL-10 receptor chain 1 (Fig. 2
, dots and asterisks) and might indicate a specific interaction with the bovine IL-10 receptor. Interestingly, the amino acid sequence of the D1701 IL-10 differs in three positions and most notably, a valine has replaced the isoleucine-115 present in all other IL-10 proteins (Fig. 2
, triangle). This isoleucine residue can be very critical for retaining stimulatory activity of human IL-10 (Ding et al., 2000
). Whether this amino acid exchange might negatively affect the IL-10 activity and contribute to the attenuation of ORFV D1701 remains speculative. Collectively, the presented results do not support the recent suggestion that ORFV, but not BPSV, encodes an IL-10 homologue (Fleming et al., 2000
). Based on the B177 IL-10 DNA sequence, a specific PCR could be used to demonstrate the presence of IL-10-specific sequences in a variety of ORFV, BPSV and PCPV (H.-J. Rziha and others, unpublished data). A possible anti-inflammatory property and suppression of an innate early immune response by the IL-10 of other PPV, as shown for ORFV NZ2 (Fleming et al., 1997
, 2000
; Imlach et al., 2002
), needs further investigation.
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ACKNOWLEDGEMENTS |
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Received 24 September 2002;
accepted 2 December 2002.