Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Author for correspondence: Geoffrey L. 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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
IFNs are soluble species-specific glycoproteins that bind to receptors on cells and trigger signal transduction leading to the expression of IFN-responsive genes and the establishment of an antiviral state (Samuel, 1991 ; Goodbourn et al., 2000
). Subsequent virus infection activates pathways leading to the inhibition of protein synthesis and hence inhibition of virus replication. IFNs are grouped according to the type of receptor to which they bind. Type I IFNs (IFN-
and -
) bind to a common receptor called the type I IFN receptor, whereas type II IFN (IFN-
) binds to the type II IFN receptor (IFN-
R). The IFN-
R is an 8590 kDa type I membrane glycoprotein and belongs to the type II cytokine receptor family (Aguet et al., 1988
; Farrar & Schreiber, 1993
). It is present on the surface of cells as a monomer but dimerizes upon binding IFN-
(Fountoulakis et al., 1991
, 1992
; Greenlund et al., 1993
) leading to signal transduction. IFN-
is a 17 kDa
-helical glycoprotein that exists as a non-covalent homodimer and its crystal structure has been determined alone (Ealick et al., 1991
; Samudzi et al., 1991
) and complexed with its receptor (Walter et al., 1995
).
Poxvirus-encoded IFN-Rs were identified first in myxoma virus: the secreted T7 protein (M-T7) bound and inhibited the biological activity of rabbit IFN-
(Upton et al., 1992
; Mossman et al., 1995b
) and was important for myxoma virus virulence in rabbits (Mossman et al., 1996
). Computational analyses showed that VV (Howard et al., 1991
), other orthopoxviruses (Massung et al., 1994
; Shchelkunov et al., 1998
), capripoxvirus (Cao et al., 1995
) and swinepox (Massung et al., 1993
) encoded related proteins. Functional studies showed that the VV, cowpox virus, camelpox virus and ectromelia virus IFN-
Rs were secreted from infected cells, bound IFN-
from a wide range of species and prevented IFN-
from binding to its cellular receptor (Alcamí & Smith, 1995
; Mossman et al., 1995a
). The broad species-specificity of the IFN-
R encoded by these orthopoxviruses was notable because cellular IFN-
Rs are usually species-specific. A surprising feature of the VV protein was its inability to inhibit mouse IFN-
although it did inhibit rat, rabbit, cow, human (Alcamí & Smith, 1995
; Mossman et al., 1995a
) and chicken (Puehler et al., 1998
) IFN-
. Nevertheless, a VV mutant lacking the B8R gene encoding the IFN-
R was attenuated in a mouse model (Verardi et al., 2001
).
Here we have studied the physical state of the VV IFN-R and report that the protein exists naturally in solution as a homodimer. Hitherto, the size of the VV IFN-
R expressed by VV strain Western Reserve (WR) or by recombinant baculovirus (AcB8R) was investigated by studying the size of the complex of the virus protein chemically cross-linked with human IFN-
after electrophoresis on SDSpolyacrylamide gels (Alcamí & Smith, 1995
; Mossman et al., 1995a
). These studies suggested that the protein from mammalian cells had a size of 43 kDa and when secreted from insect cells was 3235 kDa. The difference was attributed to the different pattern of glycosylation in mammalian and insect cells. To investigate the size of the protein further and analyse the nature of the complex formed with IFN-
, supernatants from cells infected with VV or recombinant baculovirus were run on polyacrylamide gels in the absence of reducing agent, blotted to nitrocellulose filters and probed with human 125I-IFN-
. Under these conditions 125I-IFN-
bound to proteins with an electrophoretic mobility that indicated a size between 75 and 80 kDa (data not shown). This observation suggested that the VV 125I-IFN-
R might be a dimer and this was investigated further.
Fig. 1 shows that when the supernatant from VV strain WR-infected cells was incubated with human 125I-IFN-
and protein-complexes were cross-linked chemically with EDC as described (Alcamí & Smith, 1995
) and analysed by SDSPAGE in the presence of 5%
-mercaptoethanol, three labelled bands were detected. The smaller structures (approximately 17 and 35 kDa) represent monomeric and dimeric 125I-IFN-
respectively, whereas the larger structure (about 60 kDa) represents a complex containing one molecule of the VV IFN-
R and a monomer of 125I-IFN-
, as reported previously (Alcamí & Smith, 1995
). However, when the chemically cross-linked samples were analysed by SDSPAGE in the absence of reducing agent, the 60 kDa complex was absent and was replaced by a higher molecular mass complex that had a size consistent with it containing two molecules of the VV IFN-
R and two monomers of 125I-IFN-
. Similar size complexes were formed with the IFN-
R expressed by cowpox virus and camelpox virus, indicating this seemed to be a general feature of orthopoxvirus IFN-
Rs, rather than being specific to VV. No complexes were detected when the supernatants from mock-infected cells were analysed in parallel.
|
|
The nature of the interaction between the monomeric subunits was investigated next. Soluble 35S-labelled protein produced from AcB8R-infected insect cells was mixed with Laemmli buffer that did or did not have reducing agent and was analysed by SDSPAGE. As noted previously, the protein ran as either a monomer or dimer in the presence or absence of reducing agents, respectively (Fig. 3a). To determine which conditions were able to disrupt the dimer, the protein was mixed with Laemmli buffer without reducing agent and the effect of salt or
-mercaptoethanol concentration, and pH were assessed (Fig. 3b
). Increasing the salt concentration or pH failed to disrupt the complex, and addition of reducing agent destroyed the complex completely. Collectively, these data suggest that disulphide bonds are needed to maintain the correct conformation of the complex and for dimerization to occur. However, the results do not determine whether disulphide bonds are required for correct folding of the monomeric IFN-
s that enable dimerization, or alternatively, whether direct intermolecular CC bonds mediate the formation of dimers.
|
The dimerization of orthopoxvirus IFN-Rs is interesting in comparison to the cellular IFN-
R that dimerizes only after binding the dimeric ligand. Dimerization in the latter case is needed to trigger signal transduction. The crystal structure of the extracellular domain of the IFN-
R complexed with IFN-
indicates that even after ligand binding the two IFN-
R chains remain separated by 27
(Walter et al., 1995
). Yet although the cellular and poxvirus IFN-
Rs have amino acid similarity and belong to the same superfamily, the orthopoxvirus protein exists as a dimer without ligand. The expression of dimeric IFN-
Rs by the virus may increase the neutralization capacity of the soluble IFN-
Rs. It is possible that this unique property of the VV protein is linked structurally with the ability of the protein to bind IFNs from a wide range of species, rather than being species-specific. Upon binding to its cellular receptor, IFN-
undergoes a conformational change with the flexible AB loop changing to form a 3(10)
-helix (Walter et al., 1995
), and it will be interesting to determine if a similar change accompanies binding to the virus IFN-
R. These issues require structural determination of the virus protein complexed with its ligand.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Alcamí, A. & Smith, G. L. (1992). A soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection. Cell 71, 153-167.[Medline]
Alcamí, A. & Smith, G. L. (1995). Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity. Journal of Virology 69, 4633-4639.[Abstract]
Alcamí, A., Symons, J. A., Collins, P. D., Williams, T. J. & Smith, G. L. (1998). Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus. Journal of Immunology 160, 624-633.
Alcamí, A., Khanna, A., Paul, N. L. & Smith, G. L. (1999). Vaccinia virus strains Lister, USSR and Evans express soluble and cell surface tumour necrosis factors receptor. Journal of General Virology 80, 949-959.[Abstract]
Alcamí, A., Symons, J. A. & Smith, G. L. (2000). The vaccinia soluble IFN-/
receptor binds to the cell surface and protects cells from the anti-viral effects of IFN. Journal of Virology 74, 11230-11239.
Cao, J. X., Gershon, P. D. & Black, D. N. (1995). Sequence analysis of HindIII Q2 fragment of capripoxvirus reveals a putative gene encoding a G-protein-coupled chemokine receptor homologue. Virology 209, 207-212.[Medline]
Colamonici, O. R., Domanski, P., Sweitzer, S. M., Larner, A. & Buller, R. M. L. (1995). Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon transmembrane signaling. Journal of Biological Chemistry 270, 1597415978.
Ealick, S. E., Cook, W. J., Vijay-Kumar, S., Carson, M., Nagabhushan, T. L., Trotta, P. P. & Bugg, C. E. (1991). Three-dimensional structure of recombinant human interferon-gamma. Science 252, 698-702.[Medline]
Farrar, M. A. & Schreiber, R. D. (1993). The molecular cell biology of interferon- and its receptor. Annual Review of Immunology 11, 571-611.[Medline]
Fountoulakis, M., Schlaeger, E.-J., Gentz, R., Juranville, J.-F., Manneberg, M., Ozmen, L. & Garotta, G. (1991). Purification and biochemical characterization of a soluble mouse interferon- receptor produced in insect cells. European Journal of Biochemistry 198, 441-450.[Abstract]
Fountoulakis, M., Zulauf, M., Lustig, A. & Garotta, G. (1992). Stoichiometry of interaction between interferon and its receptor. European Journal of Biochemistry 208, 781-787.[Abstract]
Goodbourn, S., Didcock, L. & Randall, R. E. (2000). Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. Journal of General Virology 81, 2341-2364.
Graham, K. A., Lalani, A. S., Macen, J. L., Ness, T. L., Barry, M., Liu, L., Lucas, A., Clark-Lewis, I., Moyer, R. W. & McFadden, G. (1997). The T1/35kDa family of poxvirus-secreted proteins bind chemokines and modulate leukocyte influx into virus-infected tissues. Virology 229, 12-24.[Medline]
Greenlund, A. C., Schreiber, R. D., Goeddel, D. V. & Pennica, D. (1993). Interferon- induces receptor dimerization in solution and on cells. Journal of Biological Chemistry 268, 18103-18110.
Howard, S. T., Chan, Y. S. & Smith, G. L. (1991). Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family. Virology 180, 633-647.[Medline]
Kotwal, G. J., Isaacs, S. N., McKenzie, R., Frank, M. M. & Moss, B. (1990). Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 250, 827-830.[Medline]
Massung, R. F., Jayarama, V. & Moyer, R. W. (1993). DNA sequence analysis of conserved and unique regions of swinepox: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue. Virology 197, 511-528.[Medline]
Massung, R. F., Liu, L. I., Qi, J., Knight, J. C., Yuran, T. E., Kerlavage, A. R., Parsons, J. M., Venter, J. C. & Esposito, J. J. (1994). Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology 201, 215-240.[Medline]
Mossman, K., Upton, C., Buller, R. M. & McFadden, G. (1995a). Species specificity of ectromelia virus and vaccinia virus interferon-gamma binding proteins. Virology 208, 762-769.[Medline]
Mossman, K., Upton, C. & McFadden, G. (1995b). The myxoma virus-soluble interferon-gamma receptor homolog, M-T7, inhibits interferon-gamma in a species-specific manner. Journal of Biological Chemistry 270, 3031-3038.
Mossman, K., Nation, P., Macen, J., Garbutt, M., Lucas, A. & McFadden, G. (1996). Myxoma virus M-T7, a secreted homolog of the interferon-gamma receptor, is a critical virulence factor for the development of myxomatosis in European rabbits. Virology 215, 17-30.[Medline]
Puehler, F., Weining, K. C., Symons, J. A., Smith, G. L. & Staeheli, P. (1998). Vaccinia virus-encoded cytokine receptor binds and neutralizes chicken interferon-gamma. Virology 248, 231-240.[Medline]
Samudzi, C. T., Burton, L. E. & Rubin, J. R. (1991). Crystal structure of recombinant rabbit interferon-gamma at 2·7- resolution. Journal of Biological Chemistry 266, 21791-21797.
Samuel, C. E. (1991). Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 183, 1-11.[Medline]
Schreiber, M., Rajarathnam, K. & McFadden, G. (1996). Myxoma virus T2 protein, a tumor necrosis factor (TNF) receptor homolog, is secreted as a monomer and dimer that each bind rabbit TNF, but the dimer is a more potent TNF inhibitor. Journal of Biological Chemistry 271, 1333313341.
Shchelkunov, S. N., Safronov, P. F., Totmenin, A. V., Petrov, N. A., Ryazankina, O. I., Gutorov, V. V. & Kotwal, G. J. (1998). The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243, 432-460.[Medline]
Smith, C. A., Smith, T. D., Smolak, P. J., Friend, D., Hagen, H., Gernart, M., Park, L., Pickup, D. J., Torrance, D., Mohler, K., Schooley, K. & Goodwin, R. G. (1997a). Poxvirus genomes encode a secreted, soluble protein that preferentially inhibits chemokine activity yet lacks sequence homology to known chemokine receptors. Virology 236, 316-327.[Medline]
Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A. & Alcamí, A. (1997b). Vaccinia virus immune evasion. Immunological Reviews 159, 137-154.[Medline]
Smith, V. P., Bryant, N. A. & Alcamí, A. (2000). Ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins. Journal of General Virology 81, 1223-1230.
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]
Symons, J. A., Alcamí, A. & Smith, G. L. (1995). Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81, 551-560.[Medline]
Upton, C., Mossman, K. & McFadden, G. (1992). Encoding of a homolog of IFN- receptor by myxoma virus. Science 258, 1369-1372.[Medline]
Verardi, P. H., Jones, L. A., Aziz, F. H., Ahmad, S. & Yilma, T. D. (2001). Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene (B8R) are attenuated in vivo without a concomitant reduction in immunogenicity. Journal of Virology 75, 11-18.
Walter, M. R., Windsor, W. T., Nagabhushan, T. L., Lundell, D. J., Lunn, C. A., Zauodny, P. J. & Narula, S. K. (1995). Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor. Nature 376, 230-235.[Medline]
Received 17 August 2001;
accepted 15 November 2001.