Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Author for correspondence: Geoffrey Smith. Present address: The WrightFleming Institute, Imperial College, School of Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK. Fax +44 207 594 3973. e-mail glsmith{at}ic.ac.uk
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Abstract |
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Introduction |
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Previously, it was demonstrated that VV strain Lister expresses a 35 kDa protein that is secreted from infected cells (Patel et al., 1990 ) and binds many CC chemokines (Graham et al., 1997
; C. A. Smith et al., 1997
; Alcamí et al., 1998
). This protein was called virus chemokine binding protein (vCKBP) (Alcamí et al., 1998
). Closely related proteins are made by some other (but not all) VV strains, cowpox virus and variola virus, and the crystal structure of the cowpox virus protein has been determined (Carfí et al., 1999
). A more distantly related protein, called T1, is encoded by Shope fibroma virus (SFV) (Upton et al., 1987
) and this also binds CC chemokines (Graham et al., 1997
). More recently, another member of this family of secreted proteins was found to be expressed by orf virus and called GIF. However, unlike the other family members which all bind chemokines, GIF binds interleukin (IL)-2 and granulocyte macrophage colony stimulating factor (GM-CSF) (Deane et al., 2000
).
Previous bioinformatic studies showed that VV encodes another protein related to SFV T1 and other members of this family (Howard et al., 1991 ). This gene was called SalF4L in VV strain WR (Howard et al., 1991
) but is called A41L using VV strain Copenhagen nomenclature (Goebel et al., 1990
). It was noted that the A41L protein had a putative signal peptide, a potential site for attachment of N-linked carbohydrate and, when aligned with the SFV T1 protein, all eight cysteine residues were conserved (Howard et al., 1991
). The gene is conserved in variola major virus strains Harvey (Aguado et al., 1992
), Bangladesh-1975 (Massung et al., 1994
) and India-1970 (Shchelkunov et al., 1994
) and the predicted protein has more than 95% amino acid identity to the VV WR A41L protein. The protein is also conserved in camelpox virus (C. Gubser & G. L. Smith, unpublished data).
The similarity of the A41L protein to vCKBP and SFV T1 suggested that it too might bind chemokines. On the basis of previous work with VV WR (which encodes A41L but not vCKBP), macrophage inflammatory protein (MIP)-1, RANTES, IL-8 and growth related oncogene (GRO)-
can be ruled out as ligands for the A41L protein (Alcamí et al., 1998
), but many more chemokines remained untested.
Here we demonstrate that gene A41L encodes a 30 kDa glycoprotein that is secreted from cells infected by all strains of orthopoxvirus examined. A VV WR mutant lacking the A41L gene replicated normally in cell culture, but was distinguishable from wild-type and revertants in vivo in two models of dermal infection. In both models, A41L expression was associated with an altered inflammatory response to VV infection. Recombinant A41L protein made by either baculovirus, yeast (Pichia pastoris) or as an Fc-fusion in mammalian cells (CHO and COS-7) was used to examine the binding of a range of chemoattractant molecules using surface plasmon resonance (BIAcore); however, to date we have been unable to identify the ligand/s for A41L.
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Methods |
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Chemokines and cytokines.
Recombinant human chemokines IL-8, neutrophil activating protein (NAP)-2, GRO-, stromal-derived factor (SDF)-1
, monokine-induced by interferon-
(MIG), interferon-
-induced protein (IP)-10, interferon-inducible T cell
chemoattractant (ITAC), RANTES, MIP-1
, macrophage/monocyte chemotactic protein (MCP)-2, eotaxin, thymus and activation regulated chemokine (TARC), liver expressed chemokine (LEC), MIP-1
, MCP-3 and recombinant murine MIP-1
, IL-2, IL-3, IL-4, GM-CSF and stem cell factor (SCF) were purchased from PeproTech. GRO-
and I-309 were purchased from R&D Systems, and the anaphylatoxin C5a was purchased from Sigma. Human pulmonary and activation regulated chemokine (PARC) and murine MIP-1
, eotaxin, MCP-1 and MIP-2 were kindly provided by Amanda Proudfoot and Tim Wells (Serono Pharmaceutical Research Institute, Geneva, Switzerland).
Construction of VV A41L deletion and revertant viruses.
A plasmid suitable for the construction of a VV strain WR mutant lacking the A41L gene by transient dominant selection (Falkner & Moss, 1990 ) was assembled by PCR and DNA cloning. Oligonucleotides 5' CCCAAGCTTGGAGGAAGGACGTAATGC and 5' CGCGGATCCAAGAATGGTCAACCACG (left flank), and 5' CGCGGATCCTCTGCAATATTGTTATCG and 5' CCCGAATTCTCATCCATTAGAGAG (right flank), from the left or right side of the A41L gene, were used together with plasmid pSalIF (Smith et al., 1991
) to generate PCR products that contained terminal HindIII and BamHI (left flank) or BamHI and EcoRI (right flank) restriction enzyme sites. These PCR fragments were digested with the appropriate restriction enzymes and cloned sequentially into plasmid pSJH7 cut with the same enzymes (Hughes et al., 1991
) to form plasmid p
A41L. This plasmid lacked 90% of the A41L ORF (codons 1201) and contained the E. coli guanylphosphoribosyltransferase gene (Ecogpt) (Boyle & Coupar, 1988
) under the control of the VV p7.5K promoter (Mackett & Smith, 1982
). The sequence of the PCR-derived regions of the plasmid was confirmed by DNA sequencing. Plasmid p
A41L was transfected into CV-1 cells infected with VV strain WR at 0·05 p.f.u. per cell and mycophenolic acid (MPA)-resistant virus was isolated by plaque assay (Falkner & Moss, 1988
). MPA-resistant virus was resolved into wild-type (vA41L) and deletion mutant viruses (v
A41L) on D980R cells in the presence of 6-thioguanine (6-TG) as described (Kerr & Smith, 1991
). These viruses were distinguished by PCR using oligonucleotides flanking the A41L gene.
A revertant virus (vA41L-rev) in which the A41L gene was re-inserted into its natural locus of vA41L was constructed by transient dominant selection using plasmid pA41L, which contained the complete A41L gene and flanking sequences cloned between the SalI and EcoRI sites of plasmid pSJH7. pA41L was transfected into v
A41L-infected CV-1 cells and MPA-resistant virus was isolated as described above. This virus was then plated onto D980R cells in the presence of 6-TG and 6-TG-resistant viruses were screened for a wild-type A41L genotype by PCR (as above).
Expression and purification of A41L protein from recombinant baculovirus.
The A41L ORF was amplified by PCR using pSalIF as template (Smith et al., 1991 ) and oligonucleotides (5' CCCAAGCTTGCAGAATGTACTCG and 5' CCGCTCGAGACAATTATCAAATTTTTTCTT) that introduced 5' and 3' HindIII and XhoI sites (underlined), respectively. The DNA fragment was digested with these enzymes and cloned into HindIII- and XhoI-cut pBAC-1 (R&D Systems) generating plasmid pAcA41Lhis so that the A41L ORF was downstream of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedron gene promoter and linked to six histidine residues at the C terminus. Plasmid pAcA41Lhis and linear AcNPV DNA (BacPAK6, Clontech) were used to construct recombinant baculoviruses pAcA41Lhis by transfection into Spodoptera frugiperda (Sf)21 cells (Alcamí et al., 1998
). To produce A41Lhis protein, Sf21 cells were infected at 10 p.f.u. per cell and at 60 h post-infection (p.i.) the supernatant was clarified by centrifugation to remove cellular debris, filtered through a 0·2 µm filter (Nalgene) and then centrifuged at 20000 r.p.m. (SW28 rotor in a Beckman L8-M ultracentrifuge) for 30 min at 4 °C to pellet virus particles. The supernatant was dialysed against 20 mM TrisHCl (pH 7·9) in 500 mM NaCl, and the A41Lhis protein was purified by Ni2+ chelate affinity chromatography followed by fast protein liquid chromatography (FPLC) using a MonoQ anion exchanger (Pharmacia). A41Lhis bound to mono-Q at pH 7·0 and was eluted in a salt gradient at 300 mM NaCl. The protein concentration was determined by the Bradford assay and by comparisons with known concentrations of BSA standards on SDSPAGE.
Immunization of rabbit to produce anti-A41L rabbit serum.
New Zealand White rabbits were injected intramuscularly with 80 µg of purified A41Lhis protein emulsified with an equal volume of Freunds complete adjuvant and boosted three times at 3 week intervals with 200 µg of the same antigen emulsified in Freunds incomplete adjuvant. Pre-immune and immune serum samples were taken prior to immunization and 2 weeks after the final immunization, respectively.
Immunoblotting.
BS-C-1 cells were infected with the indicated orthopoxviruses at 10 p.f.u. per cell for 16 h and supernatants were prepared as described (Alcamí & Smith, 1995 ). Sf21 cells were infected with AcNPV or AcA41Lhis at 10 p.f.u. per cell and 3 days later the supernatants were harvested and clarified by centrifugation (2000 g, 10 min at 4 °C). All samples were resolved by SDSPAGE on 10% gels. After electrophoretic transfer of proteins to nitrocellulose membranes (Towbin et al., 1979
), the blots were incubated sequentially with rabbit anti-A41L serum (diluted 1:2000), a goat anti-rabbit peroxidase-conjugate, and ECL reagents (Amersham) as described (Parkinson & Smith, 1994
).
Surface plasmon resonance (BIAcore) analysis.
Purified recombinant A41L or vCKBP (350 ng) was coupled to the surface of a CM5 BIAcore sensor chip. Recombinant chemoattractant molecules (e.g. cytokines, anaphylatoxin C5a, and chemokines from the CXC and CC families) were passed across the sensor chip and a positive interaction of the chemoattractant molecule (analyte) with the surface-coupled proteins (ligand) was indicated by an increase in the final base line (in response units) after the injected analyte had been eluted from the flow cells. After each injection of analyte, the sensor chip surface was regenerated with a 5 µl pulse of 0·1 M HCl, to strip the bound analyte off the ligand, and to prepare the chip surface for the next chemoattractant molecule to be injected.
Mouse intradermal model.
Groups of female BALB/c mice, between 8 and 12 weeks of age, were infected by intradermal injection into ear pinnae of 104 or 106 p.f.u. of vA41L, vA41L and vA41L-rev in 10 µl of PBS, and the diameter of lesions appearing on infected ears was estimated daily as described (Tscharke & Smith, 1999
). To measure the amount of infectious virus at the infection site, mice were sacrificed and infected ear lobes removed and frozen in 1 ml of culture medium. The whole sample was then thawed, ground with a small tapered tissue grinder (Wheaton) and subjected to two rounds of freezing and thawing followed by sonication before the titre of infectious virus was determined by plaque assay. The limit of sensitivity was 5 p.f.u. per ear lobe.
Histological examination of VV-induced lesions in a rabbit skin model.
VVs vA41L, vA41L and vA41L-rev were diluted in PBS and 104, 105 or 106 p.f.u. per site were injected subcutaneously along the flanks of a female New Zealand White rabbit, which was sacrificed 3 days p.i. Biopsy tissue samples were frozen using O.C.T. Tissue-Tek (Bayer Diagnostics) in an isopentane bath over dry ice for cryosectioning. The cryosections (10 µm thick) were placed on glass slides (Horwell Superfrost), fixed in 2% (w/v) paraformaldehyde on ice and subjected to membrane permeabilization with 0·1% Triton-X 100 in PBS. Samples were quenched at 37 °C for 5 min to remove endogenous peroxidase using glucose oxidase (0·5 units/ml) in 0·1 M phosphate buffer (pH 7·4) containing 0·2% (w/v) glucose and 1 mM sodium azide, followed by three washes with PBS (containing 0·1% Triton), and blocking for 30 min with 5% normal horse serum (Sigma). The sections were then either immunostained with mouse anti-rabbit CD43 antibody (Serotec) at a 1:100 dilution in 5% horse serum to detect infiltrating leukocytes (particularly T lymphocytes, monocytes and macrophages) or mouse MAb 5B4/2F2 against VV protein A27L (Czerny & Mahnel, 1990
) at 1:1000 dilution. Bound antibody was detected by incubation with 1:100-diluted biotinylated horse anti-mouse secondary antibody (Vector Laboratories) followed by Vectastain Elite ABC Reagent (Vector Laboratories). Immunostained sections was visualized using the chromogenic substrate diaminobenzidine tetrahydrochloride (DAB) (Sigma), which produces a reddish-brown precipitate. The sections were counterstained with cresyl violet acetate (1%), dehydrated through increasing concentrations of 70, 80, 95% and absolute ethanol, and mounted with coverslips using DPX (BDH Merck). Histological examination of these sections was done under bright field illumination using a Zeiss Axiophot photomicroscope.
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Results |
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To investigate if loss of the A41L gene affected virus growth kinetics, BS-C-1 cells were infected with vA41L, vA41L or vA41L-rev viruses at 0·01 p.f.u. per cell and the combined infectivity present in the cell and supernatant at different times p.i. was determined by plaque assay. No differences were seen between the viruses (Fig. 2a
). There were also no differences between titres of the extracellular virus fraction (data not shown). The plaque morphology formed on BS-C-1 cells by each virus was also indistinguishable (Fig. 2b
). These data indicated that despite its conservation in orthopoxviruses the A41L gene was dispensable for replication in cell culture.
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As expected, many CC chemokines (human PARC, MIP-1, MIP-1
, eotaxin, LEC, TARC, RANTES, MCP-2, MCP-3; mouse eotaxin, MIP-1
, MIP-1
, MCP-1, MCP-2) gave positive signals with vCKBP compared to A41L (data not shown), confirming they bound to vCKBP but not A41L. With the CXC chemokines IL-8, NAP-2, GRO-
, GRO-
and SDF-1 no specific binding was observed to either vCKBP or A41L. In some experiments IP-10, ITAC and MIG showed some weak binding to A41L; however, A41L-his was unable to block the migration of cells expressing CXCR3 in response to these chemokines (data not shown). Other chemoattractant molecules tested anaphylatoxin C5a and the cytokines IL-2, IL-3, IL-4, GM-CSF and SCF were not bound by A41L (data not shown).
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Discussion |
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The A41L gene is expressed early and late during infection, a pattern of gene expression that matches that of the related protein vCKBP (Alcamí et al., 1998 ), which is driven by the p7·5K promoter from VV strain WR that has been widely used in recombinant VV expression vectors (Mackett & Smith, 1982
). The A41L protein is modified by addition of N- and O-linked carbohydrate, but is still secreted from infected cells in the absence of either type of glycosylation. All orthopoxviruses tested (16 strains of VV and 2 strains of cowpox virus) expressed a soluble A41L protein and in addition the gene is highly conserved in other sequenced orthopoxviruses including several strains of variola virus and camelpox virus. Despite this conservation an A41L-deletion mutant replicated normally in cell culture.
The importance of the A41L protein was demonstrated in vivo using two different models. In a mouse intradermal model the deletion mutant induced larger lesions than wild-type and revertant controls. This was not due to differences in virus replication, since in all cases the titre of virus detectable in the ears after infection increased at the same rate and reached the same maximum titre. However, after infection with the deletion mutant, the rate at which virus was cleared was increased compared to controls. A plausible explanation is that in the absence of A41L protein, inflammatory infiltrates were increased or altered in cellular composition such that they caused more tissue damage and reduced persistence of infectious virus. Whether the reduced virus titres at late times in mice infected with vA41L were due to the direct action of the altered infiltrate, as opposed to a loss of infectable sites, enhanced virus shedding or other indirect mechanisms, remains to be shown. Further evidence that the A41L protein alters the nature of inflammatory infiltrates came from histological examination of lesions produced by wild-type, deletion and revertant viruses in a rabbit skin model. In these infected tissues there was a greater influx of CD43+ cells after infection by the deletion mutant. Moreover, with this virus the level of virus antigen was reduced at 3 days p.i. compared to controls. In contrast to the effects of the A41L protein on dermal infections, the loss of the gene did not affect the outcome of infection in an intranasal model of infection (data not shown).
The phenotype of the deletion mutant in vivo indicated that the A41L protein was having a profound affect in restricting the infiltration of at least one type of inflammatory cells into the infected lesion. Such a phenotype was consistent with the A41L protein binding and inhibiting some pro-inflammatory factor such as a chemokine or cytokine. The amino acid similarity of A41L to the VV vCKBP and the orf virus GIF protein that binds IL-2 and GM-CSF is consistent with such a view. We therefore produced recombinant A41L protein from different expression systems, coupled these proteins to the BIAcore sensor chip and sought ligands for the A41L protein using surface plasmon resonance. However, we were unsuccessful in identifying a ligand that A41L could inhibit in biological assays. As a control we used the VV vCKBP that had been expressed in the same expression systems and showed that this bound to a variety of CC chemokines as expected. The failure to identify a ligand in this way might have been due to the recombinant proteins made from baculovirus, Pichia pastoris and as an Fc-fusion protein in mammalian cells each being inactive. We consider this unlikely since the same expression systems produced biologically active vCKBP, which has a similar size, charge and overall topology to A41L. Nonetheless without a positive control for A41L binding we cannot rule out this possibility.
The A41L protein lacks a region of vCKBP that is acidic (Fig. 1b) and is exposed as an external loop in the 3-dimensional structure of the related cowpox virus protein (Carfí et al., 1999
). To determine if this region was responsible for chemokine binding by vCKBP we transferred the extra sequences into the corresponding region of A41L. However, the recombinant A41L protein was unable to bind CC chemokines (data not shown).
To continue the search for the ligand(s) for A41L we propose to undertake more detailed histological examination of infected tissue to determine what subsets of infected cells are recruited in the absence of A41L and are present when A41L has been deleted. In addition, it is possible that rather than binding a soluble factor, A41L binds to a host ligand present on a cell surface. There are precedents for soluble VV proteins binding to cells to mediate their biological function. For instance, the VV semaphorin encoded by VV gene A39R binds to the cell surface receptor plexin-1 (Comeau et al., 1998 ), the VV WR B18R protein binds to an unidentified cell surface molecule and is able to bind type I interferons in solution and at the cell surface (Alcamí et al., 2000
), and a tumour necrosis factor receptor encoded by the Lister, Evans and USSR strains of VV is able to bind to the cell surface where it can bind TNF (Alcamí et al., 1999
).
Lastly, the profound anti-inflammatory effects that A41L mediates in vivo suggest that the protein might have therapeutic application as an anti-inflammatory for acute conditions, and that the protein might reduce immune responses following infection by viruses. Therefore, the deletion of A41L from candidate poxvirus vaccines such as MVA might be beneficial in stimulating an immune response.
In summary, the A41L protein is shown to be another soluble immunomodulator secreted from VV-infected cells, but its ligand(s) has yet to be identified.
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Acknowledgments |
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Footnotes |
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c Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852, USA.
d Present address: The WrightFleming Institute, Imperial College, School of Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK.
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References |
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Received 1 May 2001;
accepted 14 June 2001.