Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Tue, 05 Apr 2016 00:41:21 Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins D. Deane, 1 N. Ueda, 2 L. M. Wise, 2 A. R. Wood, 1 A. Percival, 1 C. Jepson, 1 N. F. Inglis, 1 S. B. Fleming, 2 A. A. Mercer 2 and C. J. McInnes 1 Correspondence C. J. McInnes mcinc@mri.sari.ac.uk 1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland, UK 2 Virus Research Unit, University of Otago, Dunedin, New Zealand Received 19 August 2008 Accepted 10 December 2008 The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte– macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein. INTRODUCTION The parapoxviruses (PPVs), members of the genus Parapoxvirus of the family Poxviridae, cause a contagious pustular dermatitis in ruminants. Orf virus (ORFV) is the type species of the genus and causes disease in sheep and goats, whereas bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) affect cattle. All three viruses can infect humans. As is common amongst other poxviruses, ORFV has been shown to encode factors that either mimic host immunoregulatory proteins (Fleming et al., 1997; Haig & Fleming, 1999; Seet et al., 2003) or have the potential for interacting with components of the host immune system (Haig et al., 1998; McInnes et al., 1998, 2005; Deane et al., 2000). In doing so, it is thought that these virus-encoded factors are capable of subverting the host immune response to infection, creating an envir- onment suitable for efficient virus replication. Some of the poxvirus factors, such as the double-stranded RNA- binding protein (VAC E3L), which inhibits the antiviral effects of interferon (Haig et al., 1998; McInnes et al., 1998), are conserved across two or more poxvirus genera, whilst others appear to be genus-specific, such as the orthopoxvirus protein (VAC C21L), which inhibits the activation of the complement cascade (Isaacs et al., 1992). Some, such as the vFLIP protein (MC159) of molluscum contagiosum virus, which inhibits apoptosis, may be species-specific (Shisler & Moss, 2001; Thurau et al., 2006). ORFV has been shown to encode a protein (GIF) that is capable of binding and inhibiting both of the ovine cytokines interleukin-2 (IL-2) and granulocyte– macrophage colony-stimulating factor (GM-CSF) (Deane et al., 2000; McInnes et al., 2005). Such a protein has not been found so far in any other poxvirus; we there- fore investigated whether it was conserved in other PPVs. METHODS Viruses. ORFV strain NZ2 (Mercer et al., 2006), PCPV strains BO74 (kindly obtained from Dr Mathias Buettner, Bavarian Health and Food Safety Authority, Oberschleissheim, Germany) and VR634 (Gassmann et al., 1985) and BPSV strains V660 (Gassmann et al., 1985) and A599 (isolated de novo in the UK) were used throughout this study and were maintained by passage in primary bovine testis or fetal lamb muscle cells. Expression of the PPV GIFs. The entire open reading frames of the three PPV GIF genes were amplified by PCR and cloned individually into the pEE14 expression vector (Celltech) (Cockett et al., 1990). The integrity of the genes was verified by sequencing prior to transfection into COS-7 fibroblasts or CHO cells. Stable cell lines expressing each of the GIF proteins were established as described previously (Deane et al., 2000). Cell lines expressing the recombinant ORFV and PCPV The GenBank/EMBL/DDBJ accession numbers for the sequences of the PCPV and BPSV GIF genes are EU999744 and EU999745, respectively. Supplementary figures showing ELISA and Northern blotting results are available with the online version of this paper. Journal of General Virology (2009), 90, 970–977 DOI 10.1099/vir.0.006692-0 970 006692 G 2009 SGM Printed in Great Britain