Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK1
Author for correspondence: Antonio Alcamí. Fax +44 1223 336926. e-mail aa258{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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Molecular mimicry of cytokines and cytokine receptors is an immune evasion strategy adopted by large DNA viruses (herpesviruses and poxviruses) (Dairaghi et al., 1998 ; Nash et al., 1999
; Smith et al., 1997
; Spriggs, 1996
). Poxviruses express a unique set of secreted, soluble receptors for tumour necrosis factor, interleukin (IL)-1
, interferon (IFN)-
, IFN-
/
and chemokines, which modulate the immune response and viral pathogenesis. The genomic sequence of MCV has not revealed homologues of known orthopoxvirus cytokine receptors (Senkevich et al., 1996
). This is surprising because immune responses to MCV are weak, with little or no inflammatory reaction in the virus-filled epidermal lesions. The MCV anti-inflammatory mechanisms are not fully understood, but MCV probably encodes several other cytokine inhibitors. For example, rather than producing a chemokine-binding protein like the orthopoxviruses, MCV encodes a chemokine antagonist that may block leukocyte recruitment to sites of virus replication (Damon et al., 1998
; Krathwohl et al., 1997
).
IL-18 synergizes with co-stimulants such as IL-12 to induce IFN- production from T, B and natural killer cells (Dinarello, 1999
). Binding of this cytokine to specific cell-surface receptors induces NF-
B activation (Kojima et al., 1999
). In vivo, IL-18 is critically important for production of IFN-
and inflammatory responses, and may contribute to chronic inflammatory diseases. The production of IL-18 is important in the antiviral response since administration of this cytokine protects mice against herpes simplex virus 1 and VV infection (Fujioka et al., 1999
; Tanaka-Kataoka et al., 1999
). IL-18 is related to IL-1: the ligands share some sequence similarity, their mature forms are processed by caspase-1, their receptors are Ig superfamily members related at the sequence level, and both induce NF-
B activation (Dinarello, 1999
). Of considerable recent interest was the identification of human and mouse secreted proteins which bind IL-18 and block IFN-
induction in cells in culture and in mice injected with lipopolysacchararide (LPS) (Aizawa et al., 1999
; Novick et al., 1999
). The IL-18-binding proteins (BPs) have amino acid sequences unrelated to the IL-18 receptor and are unlikely to derive from post-translational or post-transcriptional modification of a membrane form. Intriguingly, the IL-18BPs were found to be related, at the amino acid sequence level, to a family of putative poxvirus proteins encoded by single open reading frames (ORFs) in the orthopoxviruses EV, VV, cowpox virus and variola virus (ORF D7L in variola virus Bangladesh-1975), and the suipoxvirus swinepox virus, and by three ORFs in MCV (MC51L, MC53L and MC54L) (Novick et al., 1999
; Xiang & Moss, 1999a
). A recent paper by Xiang & Moss (1999b
) reported that MCV MC53L and MC54L encode IL-18-binding activity.
Here we report that the orthopoxviruses VV, EV and cowpox virus express a soluble IL-18BP (vIL-18BP), encoded by homologues of the variola virus D7L ORF, that is secreted from infected cells and may modulate the host antiviral response. We also confirm that MCV encodes a related, larger vIL-18BP (gene MC54L) which may contribute to the lack of inflammatory response characteristic of MCV lesions.
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Methods |
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Reagents.
Recombinant human and mouse IL-18 were obtained from R&D Systems. Human IL-1 and IFN-
were from Peprotech. Human IL-18 was radioiodinated using 125I-BoltonHunter reagent (Amersham Pharmacia) to a specific activity of 2x106 c.p.m./µg.
Extraction of viral DNA and sequence of viral genes.
MCV DNA was prepared from skin lesions (Nuñez et al., 1996 ) and viral DNA from BSC-1 cells infected with EV or VV was extracted from viral cores (Esposito et al., 1981
). The vIL-18BP genes from EV strains Hampstead and Naval and VV Lister were PCR amplified with Taq DNA polymerase and oligonucleotides flanking the ORF, based on the known sequence of the gene in EV mos-3-P2 and VV WR (accession nos PXU01161 and M22812, respectively), and the PCR fragment sequenced. The MCV gene MC54L was PCR amplified with Taq DNA polymerase with oligonucleotides based on the MCV genomic sequence (Senkevich et al., 1996
), MC54L-1 (5' CCCGGATCCATGGCGCGTGGCGGAAAAGCGGG 3') and MC54L-2 (5' CCCAAGCTTCCTGACATGTCGATTCTTGGG 3'), corresponding to both ends of the ORF. The PCR fragment was cloned into pBAC-1 (Novagen) and the sequence determined in two independent clones, which were identical. Note that the 21 nucleotides at each end of the ORF correspond to the published MC54L sequence (Senkevich et al., 1996
). DNA sequencing was carried out by the DNA Sequencing Service of the Department of Biochemistry (University of Cambridge) and the sequence data were analysed using the GCG computer programs.
Expression in the baculovirus system.
Recombinant proteins, fused to a C-terminal 6xHis tag, were produced in the baculovirus system. vIL-18BP genes from orthopoxviruses were amplified by PCR with Pfu DNA polymerase using viral DNA as template. MCV genes were amplified with Taq DNA polymerase from skin samples. The oligonucleotides corresponding to the 5' and 3' ends of the ORFs provided BamHI and HindIII/PstI sites, respectively. The oligonucleotides used for EV Naval and VV Lister were P16-3 (5' CCCGGATCCATGAGAATCCTATTTCTCATCGC 3') and P16-4 (5' CCCAAGCTTGATTATATCATAAATAAAAATAG 3'), and for EV mos-3-P2 were P16-3 and P16-1 (5' CCGCTCGAGCTTCAGCCAAATATTCTTTTTTG 3'), and were based on the DNA sequence of each virus. The MC54L gene was amplified with oligonucleotides MC54L-1 and MC54L-2. DNA fragments were cloned into BamHI/PstI-digested pBAC-1 creating plasmids pNB1 (EV mos-3-P2), pNB5 (EV Naval), pNB6 (VV Lister) and pNB2 (MC54L). The DNA sequence of the inserts was confirmed not to contain mutations. The recombinant baculoviruses were produced as described (Alcamí et al., 1998a ), and named Acp11Moscow (AcNB1), Acp9Naval (AcNB5), Acp9Lister (AcNB6) and AcMC54L (AcNB2). The recombinant baculoviruses AcB15R and Ac35K have been described (Alcamí & Smith, 1992
; Alcamí et al., 1998a
). A recombinant baculovirus expressing the EV IL-1
receptor, fused to a C-terminal 6xHis tag, will be described elsewhere (V. P. Smith & A. Alcamí, unpublished). Recombinant EV IL-18BP and IL-1
receptor were purified by metal affinity chromatography by using TALON Metal Affinity Resin (Clontech).
Metabolic labelling of proteins and electrophoretic analysis.
Sf21 cells were infected with baculoviruses at high m.o.i. At the indicated time post-infection, cultures were pulse-labelled with 150 µCi/ml [35S]methionine (Amersham; 1200 Ci/mmol) and 150 µCi/ml [35S]cysteine (DuPontNew England Nuclear, 600 Ci/mmol) in methionine- and cysteine-free TC100 medium in the absence of serum. Cells or media were dissociated in sample buffer and analysed by SDSPAGE in 12% gels and fluorography with Amplify (Amersham).
Binding assays.
Supernatants from orthopoxvirus-infected BSC-1 cells or baculovirus-infected Sf cells were harvested at 2 or 3 days post-infection, respectively, and were concentrated and dialysed against PBS as described (Alcamí & Smith, 1992 ; Alcamí et al., 1998a
). Infectious VV, EV and cowpox virus present in supernatants was inactivated with 4,5',8-trimethylpsoralen and exposure to UV light (Alcamí et al., 1998a
). Binding medium was RPMI 1640 containing 20 mM HEPES (pH 7·4) and 0·1% BSA. Cross-linking to 0·4 nM 125I-IL-18 was performed in a volume of 25 µl with 5 mM bis(sulfosuccinimidyl)suberate (BS3, Pierce) as the cross-linker (Alcamí et al., 1998a
; Novick et al., 1999
). Samples were analysed by 12% acrylamide SDSPAGE.
Electrophoretic mobility shift assay.
KG-1 cells (5x106 cells in 1 ml) were stimulated for 20 min at 37 °C with 10 ng/ml of human IL-18, preincubated for 1 h at room temperature with purified vIL-18BP or control medium. Nuclear extracts were then prepared as described (Bachelerie et al., 1991 ) and incubated for 20 min at room temperature with a [
-33P]ATP-labelled probe corresponding to the NF-
B-binding element (Promega), together with poly(dI·dC). The mixtures were analysed by non-denaturing PAGE in 0·5x TBE (40 mM TrisHCl, 45 mM boric acid and 5·5 mM EDTA) and autoradiography. Binding specificity was assessed in the presence of 100-fold excess unlabelled oligonucleotide and antibodies (goat polyclonal IgG) specific for the p50 subunit of NF-
B (Santa Cruz Biotechnology).
Assay of IL-18 in murine splenocytes.
Freshly isolated splenocytes of BALB/c mice in RPMI 164010% foetal calf serum (2·5x106 cells/ml) were stimulated with 1 µg/ml LPS and 15 ng/ml murine IL-18 (Novick et al., 1999 ). IL-18 was preincubated for 2 h at room temperature with EV IL-18BP or IL-1
receptor expressed in the baculovirus system and purified by metal affinity chromatography. The production of murine IFN-
in the culture supernatant after 24 h was determined by ELISA (Diaclone Research). Purified recombinant proteins were confirmed not to interfere with the detection of IFN-
by ELISA (not shown).
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Results |
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Pulse-labelling experiments with [35S]methionine and [35S]cysteine of insect cells infected with recombinant baculoviruses showed secretion of all recombinant proteins (Fig. 3a). The apparent sizes of the vIL-18BP from EV Naval and VV Lister were smaller (9 kDa) than predicted from the sequence (14 kDa). The larger molecular mass of the EV mos-3-P2 protein (11 kDa) was consistent with the predicted C-terminal extension of the polypeptide. The 28 kDa size of the MCV protein MC54L, ranging from 26 to 29 kDa, was smaller than that reported after expression from a VV recombinant (Xiang & Moss, 1999a
), probably due to deficient glycosylation in insect cells. Binding experiments to human 125I-IL-18 followed by cross-linking showed that the recombinant proteins encoded by EV Naval, VV Lister and MCV MC54L encoded IL-18-binding activity (Fig. 3b
). Cross-linking of human 125I-IL-18 in the presence of excess unlabelled human and mouse IL-18, IL-1
and IFN-
showed binding specificity for IL-18 (not shown).
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Human IL-18 activated NF-B in KG-1 cells, as determined by electrophoretic mobility shift assay with nuclear extracts and a radiolabelled oligonucleotide of the NF-
B consensus sequence (Fig. 4a
). Binding specificity was confirmed in the presence of a 100-fold excess of unlabelled oligonucleotide, by supershift in the presence of antibodies specific for the p50 subunit of NF-
B, and by the induction of a similar band in response to IL-1
, known to activate NF-
B. Purified EV Naval vIL-18BP inhibited, in a dose dependent manner, the activation of NF-
B in response to IL-18 (Fig. 4a
).
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Discussion |
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IL-18 and IL-1, and their respective receptors, are related proteins. The binding and biological activity experiments with recombinant proteins included the orthopoxvirus IL-1 receptor, encoded by gene B15R in the VV WR strain (Alcamí & Smith, 1992
; Spriggs et al., 1992
), and showed that it does not bind IL-18. In addition, competition studies showed that natural and recombinant vIL-18BPs do not bind IL-1
. The cowpox protein CrmA and its VV homologue inhibit caspase-1 and block maturation of IL-1
(Smith et al., 1997
). Poxviruses may also inhibit IL-18 cleavage by caspase-1 within infected cells as a second mechanism to block IL-18 activity during viral infection.
VV represents a potential recombinant human vaccine. One of the best candidates is VV MVA due to its restricted host range, immunogenicity and avirulence in animal models, and excellent safety record as a smallpox vaccine. VV MVA does not express the soluble cytokine receptors found in other VV strains, with the exception of the IL-1 receptor (Antoine et al., 1998
; Blanchard et al., 1998
). Here we show that VV MVA produces IL-18-binding activity, and thus deletion of vIL-18BP from VV MVA may improve further the safety and immunogenicity of this virus vector.
The variola virus vIL-18BP has 95·2 and 93·6% amino acid identity to the active genes encoded by EV and VV, respectively. Thus, it is likely that the human pathogen which caused smallpox also utilized this immune evasion strategy. The discovery of vIL-18BP in the highly virulent EV, which causes a generalized infection known as mousepox and represents a model for human smallpox (Fenner et al., 1989 ), will allow determination of the role of vIL-18BP in vivo. These studies will also be relevant for the vIL-18BP encoded by MC54L since there is no animal model available for MCV.
The MCV MC54L polypeptide has an extended C terminus which is approximately 70 and 100 amino acids longer than the host and the orthopoxvirus IL-18BPs, respectively, and may have additional binding properties. MCV encodes two other secreted proteins, MC53L and MC51L, related to a lesser extent to IL-18BPs (Novick et al., 1999 ; Xiang & Moss, 1999a
). The MC53L ORF has recently been shown to encode IL-18-binding activity (Xiang & Moss, 1999b
) and the MC51L protein may bind other host immune molecules related to IL-18.
The expression of vIL-18BPs by MCV is particularly relevant. The complete DNA sequence of MCV revealed immune evasion strategies different from those identified in other poxviruses (Senkevich et al., 1996 ). MCV persists for months in the skin of the immunocompetent host (Moss, 1996
), and may require immune evasion molecules different to those evolved by other poxviruses that produce acute infections. The vIL-18BPs are the first soluble cytokine-binding proteins identified in MCV and may account in part for the lack of inflammatory reaction at the site of virus replication in the skin. Resolution of MCV lesions depends upon the host immune system. Thus, neutralization of MCV-encoded IL-18-binding activity constitutes a potential target for therapeutic intervention.
Poxvirus-encoded soluble cytokine inhibitors normally represent secreted versions of the binding domain of membrane-bound receptors, with the exception of two VV soluble proteins that bind type I IFNs and chemokines and have no cellular counterpart (Alcamí et al., 1998b ; Smith et al., 1998
). Interestingly, the vIL-18BPs described here have sequence similarity to a host IL-18BP distinct from IL-18 receptors, suggesting that poxviruses may have also incorporated into their genomes genes that mimic host cytokine inhibitors.
The vIL-18BP is conserved among members of the poxvirus family. We detected by cross-linking that an active protein is expressed by six out of nine strains of EV isolated from outbreaks in laboratory mouse colonies, suggesting evolutionary pressure to keep this activity in poxviruses that replicate in their natural host. The conservation of the activity in all orthopoxvirus species studied and in MCV, which does not utilize the poxvirus cytokine receptors previously identified, emphasizes the important role of IL-18 in antiviral host defence and immune regulation.
IL-18 is required in vivo for induction of IFN-, as indicated by the low levels of IFN-
produced in mice deficient in IL-18 (Takeda et al., 1998
). Therefore, the inhibition of IL-18 activity by vIL-18BP represents a novel poxvirus mechanism for inhibiting IFN-
induction, further emphasizing the importance of counteracting IFNs as an immune evasion strategy. These viruses are already known to inhibit the binding of IFNs to cellular receptors via their expression of soluble, high affinity receptors for IFN-
and IFN-
/
, and also to encode intracellular proteins that block IFN-induced antiviral pathways in the infected cell (Smith et al., 1998
).
vIL-18BP represents a novel viral immune evasion strategy and another poxvirus secreted protein that binds host immune regulatory molecules. These studies provide insights into mechanisms of viral pathogenesis and new strategies of immune modulation.
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Acknowledgments |
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Footnotes |
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References |
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Alcamí, A. & Smith, G. L. (1992). A soluble receptor for interleukin-1 encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection.Cell 71, 153-167.[Medline]
Alcamí, A., Symons, J. A., Collins, P. D., Williams, T. J. & Smith, G. L. (1998a). Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus.Journal of Immunology 160, 624-633.
Alcamí, A., Symons, J. A., Khanna, A. & Smith, G. L. (1998b). Poxviruses: capturing cytokines and chemokines.Seminars in Virology 8, 419-427.
Antoine, G., Scheiflinger, F., Dorner, F. & Falkner, F. G. (1998). The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses.Virology 244, 365-396.[Medline]
Bachelerie, F., Alcamí, J., Arenzana-Seisdedos, F. & Virelizier, J. L. (1991). HIV enhancer activity perpetuated by NF-kappa B induction on infection of monocytes.Nature 350, 709-712.[Medline]
Blanchard, T. J., Alcamí, A., Andrea, P. & Smith, G. L. (1998). Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine.Journal of General Virology 79, 1159-1167.[Abstract]
Dairaghi, D. J., Greaves, D. R. & Schall, T. J. (1998). Abduction of chemokine elements by herpesviruses.Seminars in Virology 8, 377-385.
Damon, I., Murphy, P. M. & Moss, B. (1998). Broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog.Proceedings of the National Academy of Sciences, USA 95, 6403-6407.
Dinarello, C. A. (1999). IL-18: a TH1-inducing, proinflammatory cytokine and new member of the IL-1 family.Journal of Allergy and Clinical Immunology 103, 11-24.[Medline]
Esposito, J. R., Condit, R. C. & Obijeski, J. (1981). The preparation of orthopoxvirus DNA.Journal of Virological Methods 2, 175-179.[Medline]
Fenner, F., Wittek, R. & Dumbell, K. R. (1989). The Orthopoxviruses, London: Academic Press.
Fujioka, N., Akazawa, R., Ohashi, K., Fujii, M., Ikeda, M. & Kurimoto, M. (1999). Interleukin-18 protects mice against acute herpes simplex virus type 1 infection.Journal of Virology 73, 2401-2409.
Kojima, H., Aizawa, Y., Yanai, Y., Nagaoka, K., Takeuchi, M., Ohta, T., Ikegami, H., Ikeda, M. & Kurimoto, M. (1999). An essential role for NF-kappa B in IL-18-induced IFN-gamma expression in KG-1 cells.Journal of Immunology 162, 5063-5069.
Krathwohl, M. D., Hromas, R., Brown, D. R., Broxmeyer, H. E. & Fife, K. H. (1997). Functional characterization of the CC chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2.Proceedings of the National Academy of Sciences, USA 94, 9875-9880.
Meyer, H., Ropp, S. L. & Esposito, J. J. (1997). Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses.Journal of Virological Methods 64, 217-221.[Medline]
Moratilla, M., Agromayor, M., Nuñez, A., Funes, J. M., Varas, A. J., Lopez-Estebaranz, J. L., Esteban, M. & Martin-Gallardo, A. (1997). A random DNA sequencing, computer-based approach for the generation of a gene map of molluscum contagiosum virus.Virus Genes 14, 73-80.[Medline]
Moss, B. (1996). Poxviridae: the viruses and their replication. In Fields Virology, pp. 2637-2671. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Nash, P., Barrett, J., Cao, J. X., Hota-Mitchell, S., Lalani, A. S., Everett, H., Xu, X. M., Robichaud, J., Hnatiuk, S., Ainslie, C., Seet, B. T. & McFadden, G. (1999). Immunomodulation by viruses: the myxoma virus story.Immunological Reviews 168, 103-120.[Medline]
Novick, D., Kim, S. H., Fantuzzi, G., Reznikov, L. L., Dinarello, C. A. & Rubinstein, M. (1999). Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response.Immunity 10, 127-136.[Medline]
Nuñez, A., Funes, J. M., Agromayor, M., Moratilla, M., Varas, A. J., Lopez-Estebaranz, J. L., Esteban, M. & Martin-Gallardo, A. (1996). Detection and typing of molluscum contagiosum virus in skin lesions by using a simple lysis method and polymerase chain reaction.Journal of Medical Virology 50, 342-349.[Medline]
Senkevich, T. G., Bugert, J. J., Sisler, J. R., Koonin, E. V., Darai, G. & Moss, B. (1996). Genome sequence of a human tumorigenic poxvirus: prediction of specific host responseevasion genes.Science 273, 813-816.[Abstract]
Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A. & Alcamí, A. (1997). Vaccinia virus immune evasion.Immunological Reviews 159, 137-154.[Medline]
Smith, G. L., Symons, J. A. & Alcamí, A. (1998). Poxviruses: interfering with interferon.Seminars in Virology 8, 409-418.
Spriggs, M. K. (1996). One step ahead of the game: viral immunomodulatory molecules.Annual Review of Immunology 14, 101-130.[Medline]
Spriggs, M., Hruby, D. E., Maliszewski, C. R., Pickup, D. J., Sims, J. E., Buller, R. M. L. & Vanslyke, J. (1992). Vaccinia and cowpox viruses encode a novel secreted interleukin-1 binding protein.Cell 71, 145-152.[Medline]
Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K. & Akira, S. (1998). Defective NK cell activity and Th1 response in IL-18-deficient mice.Immunity 8, 383-390.[Medline]
Tanaka-Kataoka, M., Kunikata, T., Takayama, S., Iwaki, K., Ohashi, K., Ikeda, M. & Kurimoto, M. (1999). In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection.Cytokine 11, 593-599.[Medline]
Xiang, Y. & Moss, B. (1999a). Identification of human and mouse homologs of the MC51L-53L-54L family of secreted glycoproteins encoded by the molluscum contagiosum poxvirus.Virology 257, 297-302.[Medline]
Xiang, Y. & Moss, B. (1999b). IL-18 binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins.Proceedings of the National Academy of Sciences, USA 96, 11537-11542.
Received 18 November 1999;
accepted 27 January 2000.