St Louis University Health Sciences Center, Department of Molecular Microbiology and Immunology, 1402 South Grand Blvd, St Louis, MO 63104, USA
Correspondence
R. Mark L. Buller
bullerrm{at}slu.edu
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ABSTRACT |
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INTRODUCTION |
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IL-18 plays an important role both in the innate and the specific immune response, through its potent induction of IFN- (Okamura et al., 1995
; Micallef et al., 1996
), activation of NK cell activity (Lauwerys et al., 1999
), induction of pro-inflammatory cytokines and chemokines (Puren et al., 1998
) and promotion of a Th-1 response (Takeda et al., 1998
; Dinarello, 1999a
, b
; Akira, 2000
). IL-18 has been shown to be an important mediator in the host response to many viral and bacterial infections. Treatment of mice with exogenous IL-18 confers protection in mouse models of herpes simplex virus (Fujioka et al., 1999
) and VACV infections (Tanaka-Kataoka et al., 1999
). The protective effect is likely the result of a combination of IFN-
-, NK cell- and T cell-mediated events. Mice infected with a recombinant VACV expressing IL-18 demonstrated more rapid virus clearance due to an enhanced response involving innate and specific immune components (Gherardi et al., 2003
).
Variola virus (VARV), the causative agent of smallpox, was eradicated through an intensive worldwide vaccination programme; however, the possibility of clandestine stocks in rogue nations or terrorist groups make it a potential bioweapons threat. This has led to renewed interest in understanding the pathogenesis of, and immune response to, VARV. Humans remain the only identified natural host for VARV, and while ECTV, Cowpox virus (CPXV) or VACV infections in mice have been used to develop a greater understanding of orthopoxvirus pathogenesis and immunity, these do not typically cause disease in humans. Thus, VARV is uniquely adapted to infect, replicate and cause disease in humans, and its immunomodulatory genes may be uniquely adapted to their cognate human ligands. In this study, we have demonstrated that VARV encodes a functional IL-18BP, D7L. The affinity of D7L for human and murine IL-18 was compared with that of the ECTV IL-18BP, p13. Furthermore, we have demonstrated that D7L is capable of interacting with glycosaminoglycans (GAGs) via residues in the C terminus, while p13 lacks this function. Importantly, D7L can interact with both GAG and IL-18 simultaneously, indicating that the binding sites are distinct.
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METHODS |
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Cloning.
The ECTV p13Fc (human IgG1) fusion gene in the expression vector pDC409 was a generous gift from Amgen. A 10·4 kbp amplicon, representing approximately 5 % of the genome of VARV (strain Bangladesh-1975), was obtained from the Centers for Disease Control, Atlanta, GA, USA. In agreement with World Health Organization regulations, all use of VARV DNA was limited to situations where accidental recombination with other poxvirus DNA could not occur. D7L was amplified by PCR using the following primers: BSH-D7L5': 5'-gcgttcatagtcgacatgagaatcctatttctc-3' and BSH-D7L-Fc: 5'-agagatctctttagctttagccaaatattc-3'. Primer BSH-D7L-Fc was used to generate an Fc-tagged fusion protein. The D7L PCR product was ligated into pDC409, generating pDC409-D7L-Fc. The sequence was verified on both strands.
The 5' sequence of D7L was mutated using a site-directed mutagenesis kit (Genetailor; Invitrogen). Primers were designed to replace the C-terminal residues of D7L (KKNIWLK) with the sequence present in p13 (KKEYLAE): 5'-ggcgtttcaaaaaaggaatatttggctgaaagatcttgt-3' and 5'-cttttttgaaacgccatttagcgtagt-3'. The mutagenic PCR and bacterial transformation were performed according to the manufacturer's directions. The sequence was verified on both strands.
Protein expression and purification.
CV-1/EBNA-1 cells were transfected with plasmids containing the Fc fusion constructs using a DEAEdextran method. Briefly, DNA was mixed with complete cell culture medium containing chloroquine and then complexed to DEAEdextran. The complexes were added to a T-150 flask containing complete medium with chloroquine and incubated for 4·5 h at 37 °C, 5 % CO2. The medium was then replaced with DMEM containing 10 % DMSO for 5 min at room temperature and finally replaced with DMEM-10. Cell culture supernatants were collected weekly for up to 4 weeks and stored at 20 °C. Expression was assessed by Western blotting. Cell supernatants were then pooled and purified by FPLC (AKTA; Amersham Pharmacia Biotech) on a 5 ml HiTrap Protein-G column. The column was equilibrated and washed with PBS, pH 7·4, and bound protein was eluted using 50 mM citrate, pH 3. The column was washed with 50 mM glycine, pH 2, to remove any remaining proteins. Fractions were collected, neutralized and assessed by silver staining (Bio-Rad). Peak fractions were pooled and dialysed against PBS, pH 7·4, and filter-sterilized using a 0·22 µm syringe filter. Purity was assessed by analysing samples by 12 % SDS-PAGE followed by silver staining. Protein concentration was determined by the Lowry method. In all experiments, full-length purified Fc fusion proteins were used.
Western blotting.
Expression and purification of p13Fc, D7LFc and D7L-KKEYLAE was assessed by SDS-PAGE followed by Western blotting. PVDF membranes were blocked in 5 % non-fat dried milk in PBS and incubated with rabbit anti-p13 antiserum, which is cross-reactive with D7L. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (KPL) was used as a secondary antibody and was detected using ECL-Plus (Amersham Biosciences) and exposed to film or captured by digital imaging (Alpha Innotech). Human IL-18 was detected using anti-huIL-18 rabbit polyclonal antibody (Cell Sciences) and HRP-conjugated secondary antibody as above.
IL-18 bioassay.
KG-1 cells were precultured at 2x105 cells ml1 for 4 days before use in the bioassay. Cells were centrifuged and resuspended at a concentration of 3x106 cells ml1 in medium containing 20 ng TNF- ml1 (R&D Systems). HuIL-18 at 10 ng ml1 was mixed with medium or with dilutions of D7LFc or p13Fc. The IL-18/IL-18BP mixture was incubated for 1 h at 37 °C and then 100 µl cells were added and incubated for 24 h in a 96-well plate. The supernatants were harvested, centrifuged and stored at 20 °C until they were analysed for IFN-
levels. The supernatant was assayed in an ELISA to detect IFN-
, according to the manufacturer's recommendations (BD Pharmingen).
Surface plasmon resonance.
Interaction of the binding proteins with IL-18 was measured by surface plasmon resonance (SPR) on a BIAcore 2000 (Biacore Inc.). All flow cells (FCs) of a CM5 chip were activated with EDC/NHS for 6 min at 5 µl min1. Anti-human IgG (Jackson Immunoresearch) was immobilized on all FCs, using a flow rate of 5 µl min1, for 7 min. The surface was blocked by injecting ethanolamine for 7 min at 5 µl min1. For kinetic studies, Fc fusion proteins were captured in FC2 or FC3, using FC1 as the reference cell, followed by injection of IL-18 at varying concentrations. Capture levels were kept low to minimize the effects of mass transfer. Cycle conditions were as follows: 7 min capture at 5 µl min1, 3 min analyte injection at 30 µl min1 over all FCs, followed by 10 min dissociation time. The surface was regenerated with a 1 min injection of 5 mM HCl, pH 1·5. At least one of the dilutions was run in duplicate in each experiment and IL-18 concentrations were injected in random order. The experimental Rmax was typically 60 % of the theoretical, indicating that a fraction of the captured protein was inactive or inaccessible for interaction with the analyte. To rule out the possibility of leaching between FCs during association and dissociation phases, experiments were repeated with protein captured on different FCs or individually on FC2. All SPR data were analysed and fitted globally using BIAevaluation 3.2 software.
For SPR analysis of interaction with heparin, a streptavidin (SA) sensor chip (Biacore Inc.) was used to capture biotinylated BSA on FC1 and biotinylated BSAheparin on FC2 (both from Sigma). Analyte was injected at 30 µl min1 at varying concentrations for 3 min, followed by 10 min dissociation. The surface was regenerated with a 15 s injection of 1 M NaCl in 50 mM NaOH at 5 µl min1.
A GAG competition assay was performed to measure the relative affinity of D7L for different GAGs. A constant amount of D7L was pre-incubated with increasing concentrations (2·5 µg ml1, 25 µg ml1, 0·25 mg ml1 and 2·5 mg ml1) of soluble heparin, heparin sulphate (HS), chondroitin sulphate B (CSB) (all three derived from porcine intestinal mucosa) or chondroitin sulphate A (CSA) (from bovine trachea; Sigma). Samples were then injected over the BSAheparin surface and BSA alone reference surface. The maximum response was measured 20 s before the end of injection.
HeparinSepharose pull-down assay.
HeparinSepharose beads (Amersham Biosciences) were washed with HEPES buffered saline and resuspended in the same buffer to generate a 50 % slurry. Beads were mixed with 130 nM protein and incubated at 4 °C for 2 h. Immediately prior to incubation an aliquot was removed for later analysis as the input fraction. The beads were recovered by centrifugation and the supernatants were collected for later analysis (unbound fraction). The bead fraction was washed three times and analysed for the presence of IL-18BP or used in a subsequent incubation with huIL-18 for 2 h at 4 °C. Beads were recovered as before and the unbound fraction collected. Samples were analysed for IL-18BP or IL-18 by Western blotting. A competition assay was performed by pre-incubating D7LFc with 2·5 mg soluble heparin ml1 for 2 h followed by incubation with heparinSepharose beads as described above.
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RESULTS |
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DISCUSSION |
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Orthopoxvirus immunomodulatory proteins frequently display broad species specificity (Mossman et al., 1995; Alcami & Smith, 1995
, 1996
; Symons et al., 1995
; Smith & Alcami, 2000
). Orthopoxviruses may have had multiple maintenance hosts throughout their evolution, resulting in diversification of ligand binding capability. Similarly, the IL-18BPs can bind multiple species of IL-18. Interestingly, both D7L and p13 demonstrated higher affinity for muIL-18 than huIL-18. The affinity for muIL-18 is comparable with that of the high-affinity (0·4 nM) IL-18 receptor (Yoshimoto et al., 1998
; Debets et al., 2000
) found on the cell surface, suggesting that p13 and D7L could effectively compete against the receptor for muIL-18. The affinity for huIL-18 is significantly lower and is in the range of the low-affinity (1131 nM) cellular receptor (Yoshimoto et al., 1998
). Inspection of the association and dissociation rates of p13 and D7L reveals that the difference in binding affinities for the mouse and human ligands is due to a faster association rate for murine IL-18. Therefore, the binding protein and muIL-18 complex is formed more rapidly; however, once formed the complex with either ligand is equally stable. It is important to note that huIL-18BP and MC54L also display an approximately fivefold higher affinity for muIL-18 than huIL-18 (Xiang & Moss, 1999b
) indicating that the species preference is common among the IL-18BP family. The higher affinity for muIL-18 in these proteins is also primarily due to a faster association rate. The higher-affinity binding may be due to an intrinsic property of muIL-18 that allows better binding.
Recently, the smallpox inhibitor of complement enzymes, SPICE, was shown to be significantly more potent at inactivating human complement than its homologue in VACV (Rosengard et al., 2002). The VARV and VACV complement inhibitors differ by less than 5 %, suggesting that minor changes can significantly change species specificity. The current study demonstrates that VARV immune modulators are not necessarily better suited for human ligands and that adaptations overcoming human immunity may not explain the narrow host range or considerable pathogenicity of VACV. The species specificity of CPXV and ECTV IL-18BPs has been assessed and has correlated with the host range of the pathogens (Smith et al., 2000
; Calderara et al., 2001
). These studies demonstrate that, despite the fact that VARV is a human pathogen, D7L has a lower affinity for the ligand from its natural host, as does the IL-18BP of another obligate human pathogen, MOCV (Xiang & Moss, 1999b
). Therefore, it is possible that higher-affinity binding of huIL-18 is not possible since huIL-18BP and IL-18BPs from two human pathogens demonstrate higher-affinity binding for muIL-18. Thus these proteins may already possess optimal binding within the limitations of the structure of the IL-18BP family.
GAGs are an abundant component of the extracellular matrix and cell surface and are composed of repeating disaccharide units, usually modified with sulphyl groups or alkyl groups, attached to a protein core. An enormous diversity of structures can be formed based on variations in the disaccharide backbone and modifications and differential tissue expression results in a complex and unique extracellular environment. GAGs participate in a variety of processes including binding of growth factors and chemokines, which contributes to protein function by helping to establish a solid-phase gradient, maintain a high local concentration or aid in association of a ligand with its receptor (Schonherr & Hausser, 2000). Viral proteins such as the Myxoma virus chemokine binding protein, M-T1 (Seet et al., 2001
), and MC54L, the MOCV IL-18BP (Xiang & Moss, 2003
), can also bind GAGs. Since there are several examples of secreted immunomodulatory poxvirus proteins that bind GAGs, it is likely that this activity is important in maximizing the ability of the proteins to compete with the cellular receptor by maintaining high concentrations at the site of infection. In this study we have demonstrated that the VARV IL-18BP can also interact with GAGs in vitro. D7L has an appreciable affinity for heparin (56 nM); however, p13 does not demonstrate detectable binding. For comparison, MC54L binds heparin with an affinity that is two orders of magnitude higher than D7L due to the longer, heavily charged C terminus (Xiang & Moss, 2003
).
GAG binding is conferred primarily, but not exclusively, through ionic interactions with positively charged residues (Mulloy & Linhardt, 2001). We have provided evidence that the region involved in binding to GAGs is the C-terminal sequence present in D7L but not p13, and that the binding site is physically distinct from the IL-18 binding site, since both ligands can be bound simultaneously. The C terminus of D7L contains three lysines that closely fit a common heparin-binding motif, and although p13 contains two of these lysines, acidic residues are also present. Interaction with GAGs is primarily driven by ionic interactions with positively charged amino acids; thus the presence of negative charges is likely to interfere with binding.
In a competition assay, D7L bound heparin best, HS and CSB equally but less than heparin, and CSB with the lowest affinity. Although specific conclusions on the components of GAGs that may be involved in binding cannot be made, some structures are suggested based on the relative affinities for each. Binding may be dependent on the degree of sulphation, with the most highly sulphated GAG, heparin, binding best. In addition, unlike the other GAGs tested, CSA does not contain L-iduronic acid, indicating that this may also contribute to binding.
At this time, it is only possible to speculate why D7L, but not p13, binds GAGs. The maintenance hosts of ECTV and VARV differ, indicating that this function may be more important in the human host. The IL-18BP of MOCV, a human pathogen, also binds GAGs via residues in the C terminus (Xiang & Moss, 2003). However, analysis of the nucleotide sequences of D7L and p13 reveals that the different C termini are the result of a single nucleotide deletion, resulting in a frame shift and later termination in p13. Furthermore, the C terminus of p13 differs among ECTV strains; p13 of ECTV Moscow (used in this study) and Hampstead are identical, while other strains, including ECTV Naval, have a C terminus identical to D7L. The CPXV IL-18BP C terminus is unique among orthopoxviruses and is likely to lack GAG binding activity, while VACV and Monkeypox virus encode IL-18BPs with D7L-like C termini. Thus it is difficult to conclude whether GAG binding is a gain-of-function mutation or whether some viruses have lost this function during their evolutionary history.
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ACKNOWLEDGEMENTS |
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Received 13 December 2003;
accepted 9 February 2004.