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. 2000 Feb;74(3):1313-20.
doi: 10.1128/jvi.74.3.1313-1320.2000.

Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2

D Deane  1 , C J McInnes, A Percival, A Wood, J Thomson, A Lear, J Gilray, S Fleming, A Mercer, D Haig
Affiliations

Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2

D Deane et al. J Virol. 2000 Feb.

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 K(d) of 369 pM and ovine IL-2 with a K(d) 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.

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Figures

FIG. 1
FIG. 1
DNA sequence of, and predicted amino acid sequence encoded by, the NZ2 orf virus GIF cDNA. The entire GIF ORF together with 100 bases of flanking sequence on either side of the ORF is shown. The initiator methionine is 20.1 kb from the right terminus of the orf virus genome. The gene is transcribed toward this terminus. The mature secreted protein starts with Ala20. The region containing potential transcription promoter and/or initiator sequences is underlined.
FIG. 2
FIG. 2
Expression of recombinant and nonrecombinant GIF in virus-infected FLM cells. (A) Silver stain of recombinant GIF produced in CHO cells and purified by GM-CSF affinity chromatography followed by Sephacryl S-200 gel filtration. The positions of molecular mass markers are shown on the left. (B) Northern analysis of GIF mRNA expression in FLM cells at 18 h after infection with NZ2 virus at an MOI of 20 TCID50 in the presence (lane 2) or absence (lane 1) of CA. Positions of RNA size markers (in bases) are indicated. (C) GIF expression in FLM CFSs at various times after infection with NZ2 (MOI = 2 TCID50) in the absence of CA. Shown is a Western blot, prepared with rabbit anti-GIF, of samples subjected to SDS–15% PAGE. Recombinant GIF and GM2 (the product of an uncharacterized ORF adjacent to the GIF gene) were included as a positive and negative control, respectively. Both the recombinant and native GIFs are 28-kDa proteins.
FIG. 3
FIG. 3
GIF forms dimers and tetramers under physiological conditions. (A) Separation of 125I-GIF on a Sephacryl S-200 column in 0.15% (vol/vol) CHAPS buffer at pH 7.4. Each fraction contained 1 ml of eluate. Two peaks of GIF eluted, with apparent molecular masses of ∼112 and ∼56 kDa. The arrows indicate the elution positions of protein standards (β-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin, and chymotrypsinogen A) used to construct the molecular mass-versus-elution volume standard curve. (B) ovGM-CSF–GIF ligand blot showing the pooled and concentrated (10-fold) Sephacryl S-200 fractions of 125I-GIF that bound to ovGM-CSF (19–21-kDa dimer), subjected to SDS–15% PAGE under nonreducing conditions and electrotransferred onto nitrocellulose strips. (C) Pooled and concentrated 125I-GIF fractions from the S-200 column, subjected to SDS–15% PAGE under nonreducing conditions and revealed by autoradiography. Fractions 12 to 18 contained the 112-kDa GIF peak from the S-200 column, and fractions 33 to 38 were from the 56-kDa peak. All fractions showed a 28-kDa protein band. To the left of panels B and C are shown the positions of molecular mass markers.
FIG. 4
FIG. 4
GIF binding of ovGM-CSF and ovIL-2: 125I-GIF–cytokine ligand blot analysis. 125I-GIF was used to probe a range of ovine cytokines (A) and ovine, human, and murine cytokines (B) which were separated by SDS–15% PAGE and transferred to nitrocellulose membranes. 125I-GIF bound to ovGM-CSF and ovIL-2. As a specificity control, 100 nM of unlabelled GIF was used to compete for 125I-GIF binding to the cytokines. This inhibited 125I-GIF binding to ovGM-CSF and ovIL-2. The positions of molecular mass markers are indicated.
FIG. 5
FIG. 5
GIF inhibits ovGM-CSF and ovIL-2 activities. (a) GIF inhibition of ovGM-CSF activity in the soft-agar bone marrow cell clonogenic assay. Colonies in triplicate cultures were counted on day 14 of culture, and the numbers were adjusted to represent the mean number of colonies, ± the standard error of the mean, that developed from 105 bone marrow cells plated on day zero. ovGM-CSF was used at 460 pg/ml; ovIL-3 and GIF were used at 520 and 50 ng/ml, respectively, based on dose-response experiments. (b) GIF inhibition of ovIL-2 activity in the 48-h T-cell proliferation assay. C.P.M., mean counts per minute of [3H]thymidine incorporated into dividing cells in quadruplicate wells ± standard error of the mean. ovIL-2 and GIF were used at 40 and 100 ng/ml, respectively, based on dose-response experiments. ∗, P < 0.002 versus ovGM-CSF-stimulated colonies, (a) or IL-2-stimulated T-cell proliferation counts per minute (b).
FIG. 6
FIG. 6
GIF and ovGM-CSF in virus reinfection and control afferent lymph plasma. (a) GM-CSF concentrations in afferent lymph plasma samples from reinfected and control sheep. Control sheep (no. 18 and 26) received UV-inactivated virus intradermally. (b) Detection of GIF, by ovGM-CSF inhibition ELISA, in afferent lymph plasma samples obtained at various times after orf virus reinfection of sheep 15, 25, and 34 on day zero. Lymph plasma samples were spiked with 8 ng of GM-CSF/ml (4 ng/ml for sheep 15 samples, hence the lower OD) and analyzed for GM-CSF by ELISA. Rabbit anti-GIF was used as a specificity control to neutralize GIF in the assay.
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