Cytokine receptors encoded by poxviruses: a lesson in cytokine biology

Cytokinereceptorsencodedby poxviruses: a lessonin cytokinebiology Antonio Alcami and Geoffrey L. Smith Poxviruses encode soluble Llersions of cytokine receptors, which are secreted from the infected cell to block the activity of the cognate cytokine. These viruses offer a unique model system to study the contribution of cytokines to the host response against infection. As discussed here by Antonio Alcami and Geoffrey Smith, characterization of poxvirus proteins that counteract the immune response may lead to the identification of novel cytokines or cytokine receptors, as well as novel strategies to modulate the inflammatory response. Poxviruses have played important roles during the history of medicine ‘J . In 1798, Edward jenner introduced cowpox virus as the first vaccine for the prevention of a disease, in this case smallpox, a devastating scourge of humanity. In the 20th century, vaccinia virus was used to combat smallpox leading, in 1977, to the first successful global eradication of an infectious disease. Poxviruses have also played an important role in microbiological research2,;. Vaccinia virus was the first mammalian virus to be observed microscopically, as well as the first to be grown in tissue culture, physically purified and biochemically characterized. Studies on vaccinia revealed that virus particles may contain enzymes for RNA transcription, and these have provided important models to dissect mRNA synthesis. The dcvelopment of molecular techniques for insertion of foreign genes into the virus genome opened the possibility of using recombinants of vaccinia virus as vaccines against other diseases, and provided researchers with an important laboratory tool for molecular characterization of the expressed proteins. Once again, poxviruses are emerging as a model to study biological processes. The discovery that poxviruses possess a fascinating machinery to counteract the host immune response is revealing fundamental information concerning the mechanisms of viral pathogenesis, and is providing excellent research tools to study the complex immune system. Poxvirus strategies for immune evasion Poxviruses are complex cytoplasmic viruses (Fig. 1), with a large double-stranded DNA genome that has been sequenced and shown to accommodate approximately 200 genes 4. During evolution, poxviruses have acquired an impressive variety of genes encoding proteins that counteract the host response to infection, as summarized in Fig. 2 and reviewed elsewhere’-‘I’. Most of these proteins deal with nonspecific immune mechanisms, such as the complement system, interferons (IFNs) and the inflammatory response, which are rapidly induced and constitute the first host response against the invading organism. Many of these poxvirus proteins have sequence similarity with host immune factors,

suggesting that poxviruses have ‘stolen’ and modified host genes in horder to inhibit the activity of key immune modulators. Although other viruses have defensive strategies, the repertoire of homologues of host immune proteins displayed by poxviruses is without precedent. A possible reason for this is the infectious cycle of poxviruses. Other viruses establish more-sophisticated relationships with their host, such as latency, that enable escape from the immune system and persistence in the host for long periods of time. By contrast, poxviruses produce a cytolytic infection, replicating in the presence of an active immune system and, thus, may need more weapons to combat host defences. Cytokines regulate the inflammatory and immune response, and may induce an antiviral state in the cell (i.e. via IFNs) or destroy virus-infected cells [i.e. via tumour necrosis factor (TNF)]. Thus, cytokines constitute good targets for virus evasion strategies. Some poxvirus genes encode homologues of the extracellular binding domain of cellular cytokine receptors, lacking the transmembrane and cytoplasmic domains of their cellular counterpart. These proteins are secreted, bind the cognate cytokine and interfere with its activity by preventing interaction with cellular receptors (Table 1). Other viruses have also evolved mechanisms to counteract the activity of cytokines. Herpesviruses encode receptors for chemokines, which are potent chemoattractants of immune cells. In contrast with the poxvirus soluble receptors, these chemokine receptors are present on the surface of the infected cell and probably function in a different way27. A gene predicted to encode a similar chemokine receptor has been found in swinepox virus, but to date no functional data have been reported”. Epstein-Barr virus (EBV) encodes a homologue of the host cytokine interleukin 10 (IL-lo), which suppresses aspects of the cellular immune response’x. Adenoviruses counteract the activity of cytokines such as TNF with intracellular proteins that show no amino acid similarity to known cellular factors29. The finding that poxviruses encode soluble receptors for cytokines such as TNF, IL-lB, IFN-y and IFN-a/P (Table 1) emphasizes the importance of these cytokines

in the immune response. This is corroborated bv the existence of complementary intracellular mechanisms encoded by poxviruses to inhibit the IFN-mediated responses and the maturation of IL-lp (Fig. 2). This article describes how the characterization of poxvirus cvtokine receptors, and their mode of action, is revealing physiological functions of cytokines and giving valuable information leading to a better understanding of the immune system. Function of cytokines in vivo Some properties of the poxvirus cytokine receptors have been unexpected, and this may reflect our inadequate understanding of the functions of some cytokines. The vaccinia IL-lp receptor (IL-1PR) best illustrates this point. There are two host IL-IRS (type I and II) and three species of IL-l: IL-la, IL-lfi and IL-1 receptor antagonist (IL-lra). IL-la and IL-lb induce IL.-l mediated responses, while IL-lra is a natural inhibitor that binds to the cellular receptor but does not trigger a signa130. The poxvirus receptor binds IL-ID with high affinity, but does not bind IL-la or IL-lra (Ref. 21). This suggests that, although IL-la and IL-lfi seem to play a similar role in the immune response in some experimental models 3o, IL-lb may be more important against virus infections. The failure of the virus receptor to bind IL-lra prevents interference with the host’s natural inhibitor. Expression of the vaccinia IL-1PR prevents the onset of fever in mice infected with vaccinia virus (A. Alcami and G.L. Smith, unpublished). This appears to be the first example of a virus mechanism that blocks the febrile response, one of the major IL-l-mediated systemic effects that contributes to host defence”‘. Again, this result is unexpected since other cytokines, such as IL-la, TNF, IL-6 and IFN-y, are thought to mediate fever. This suggests a central role for IL-1p as a mediator of fever in virus infections. Analysis of the function of cytokines in vivo is often difficult because of the lack of appropriate models. Inoculation of recombinant cytokines or inhibitors may not provide physiological doses, and the proteins may not last for long periods or reach the appropriate site of action. By contrast, replication of vaccinia virus induces the immune response naturally and, simultaneously, delivers continual large amounts of the inhibitor both at the site of infection and systemically, thus representing an ideal model system. Similarly, expression of recombinant cytokines by vaccinia virus has been used as an experimental approach to evaluate their role against virus infections”‘. Strategies for cytokine blockade and inhibition of inflammation The activity of IL-l may be neutralized by soluble IL-1Rs or IL-lra. The acquisition of soluble receptors by poxviruses suggests that these may be more potent as anti-inflammatory reagents. Recent studies suggest that the cellular type II IL-1R does not transduce signals; rather, it is shed from the cell surface and acts (like the virus counterpart) as a decoy receptor to inhibit IL-1 activity3”. Interestingly, the soluble cellular type II IL-1R has a higher affinity for IL-lp than for

Fig. 1. Electron micrograph of a vaccinia-infected cell showing virus particles bemg transported tu the plasma membrane and released into the medium. Magnification = X 120 000. Modified figure reproduced u,lth pcrmlssmn from Ref. 39.

IL-ltu or IL-lra. This similarity to the virus IL-1PR would be consistent with IL-lp being the major secreted proinflammatory form of IL-I. The use of soluble receptors specific for IL-lp in therapy may have the advantage of not interfering with 11.-1~~or IL-lra. Along the same line, release of soluble versions of host TNFRs and IFN-yRs similar to those encoded by poxviruses may constitute a host mechanism to control the action of these cytokines’“. Natural hosts of poxviruses The species specificity of the poxvirus cytokine receptors may indicate the natural hosts of these viruses. For example, the TNFR encoded by myxoma virus, a pathogen of rabbits, specifically binds rabbit but not human or mouse TNF (Ref. 35). These studies are particularly relevant for vaccinia virus, since its origin and natural host(s) are unknown’.‘. Cowpox virus was introduced 200 years ago as a smallpox vaccine, but all the vaccines used this century have been derived from a different species, not presently found in nature, that was named after the vaccination procedure - ‘vaccinia

t

VGF

IFN-mediated response

(CllR) r

3&HSD

(E3L, K3L)

. .

/

b4.W

Blockage of MHC class I presentation to CTLs

prdL-1 f3

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Fig. 2. Immune evasion mechanisms encoded by poxviruses. The virus proteins that block diff erent arms of the host response to infection (complement activation, cytokine function, antigen presentation to CTLs, inflammation) or mimic host growth factors are represented. For details see Refs J-10. The origin of the arrows indicates the localization of proteins: intracellular, plasma membrane or extracellular. Nomenclature for vaccinia virus genes ts from strum Copenhagen, except for the BlSR and B18R genes, which are from the Western Reserve strain; in those cases where the gene is not present in vaccinia virus, the myxoma virus (MV), swinepox virus (SPV) or orf virus (OV) genes are indrcated. The function of all the proteins has been demonstrated, except for the IL-IR-like protein, the VEGF-ltke protein and the BSR protean related to complement control protetns (possible functions for these proteins are based on sequence similarity). No protein has been identified to be responsible for the blockage of antigen presentation to CTLs. Virus proteins that block inflammation andlor contribute to vzrus virulence but have no similarities with current databases, and interact with as yet unidentified ligands, are also represented. Abbreviations: 2’5’oligo A, L’Soligoadenylate; 2 ‘S’OS, 2’5’oligo A synthase; 3/3-HSD, 3P-hydroxysteroid dehydrogenase; CTLs, cytotoxic T lymphocytes; dsRNA, double-stranded RNA; elF2cq eukaryotic initiation factor 2a; ICE, IL-l fl-conuerting enzyme: EN, rnterferon; FN-c@R, FN-(Yip receptor; IL-l& interleukin 1p; IL-1 /JR, IL- I p receptor; MHC, major histocompatibility complex; PKR, dsRNA-dependent protein kinase; serpin, serin protease inhibitor; TNFR, tumour necrosis factor receptor; VCP, vaccinia complement control protein: VEGF, vascular endothellal growth factor: VGF, vaccinza growth factor.

virus’. Because of the high species specificity of the IFN system, the IFNqR is a good choice for these studies. The vaccinia and cowpox IFN-yRs bind and block the biological activity of human, bovine and rat, but not mouse, IFNq (Ref. 25). In a parallel study, it was found that the vaccinia IFN-yR binds human and rabbit, but not mouse, IFN-y, while the myxoma IFNqR is specific for rabbit IFN-7 (Refs 24,36). This novel, broad species specificity of the vaccinia and cowpox IFN-?R may have provided an evolutionary advantage by aiding virus replication in several host species. A similar broad species specificity has been shown for the vaccinia IFN-eJPR (Ref. 26).

Soluble cytokine receptors and virus virulence Virus mutants in which cytokine receptor genes have been disrupted or deleted have been tested in animal models of infection and compared with wild-type virus. This provides a perfect natural model to address the contribution of cytokines to the host immune response. Since expression of cytokine inhibitors may help the virus to evade host defences, inactivation of the genes is expected to decrease virus virulence. Consistent with this idea, deletion of the myxoma TNFR or the vaccinia IFN-a&R attenuated the infection in animal models, demonstrating an important role for TNF and IFN-o(/P against poxviruses12,2”.

Inactivation of the IL-ll3R from vaccinia virus has produced contradictory effects in mice, decreasing virus virulence after intracranial infection20 but enhancing virus virulence when administered intranasally?‘. However, this apparent inconsistency may be in keeping with the biological properties of IL-l: this cytokine induces beneficial effects in the host, potentiating the inflammatory and immune responses but, when produced in large amounts, contributes to the pathological process 30. Since a natural route of poxvirus infections is the respiratory tract2, the IL-ll3R may function to prevent the detrimental effects of excessive IL-ll3, consistent with the finding that the blockade of IL-l reduces the severity of various diseasesj’. This strategy will increase host survival and give the virus more time to replicate before the host expires. Thus, viral pathogenesis is the result of a complex interaction between virus and host factors, with some virus proteins acting to reduce the damage that the immune system can cause to the host. Cytokines interact with each other at different levels inside a complicated network, thus the contribution of particular cytokine inhibitors to virus virulence may vary depending on other virus immune modulators being expressed in the same virus. A particular combination of virus proteins will be responsible for the final virulence phenotype. Smallpox Variola virus was one of the most virulent poxviruses that, until its eradication, produced smallpox, with case-fatality rates of up to 40% (Refs 1,2). The complete DNA sequence of the variola virus strains Bangladesh-1975 and India-1967, and a region of the Harvey strain, have been reported. Interestingly, not all of the poxvirus cytokine receptors are predicted to be active in variola virus (Table l), although the genes for IFN-(Y&R, IFN-?R and TNFR are predicted to encode active proteins. However, a second TNFR gene is

shown to be deleted from the genome. The reasons for the acquisition of two TNFR genes by some viruses are not yet understood, but the inactivation of one of them in variola virus suggests different roles in viral pathogenesis. Surprisingly, the IL-ll3R gene is inactivated by mutations in variola virus, which infected through the respiratory tract. This correlates with an increased virulence of vaccinia virus lacking the IL-ll3R when inoculated intranasally”. The World Health Organization is considering the destruction of the remaining stocks of variola virus, held at the Center for Disease Control in Atlanta and at the Institute for Viral Preparations in Moscow. Although DNA fragments of the virus genome would still be available, the destruction of the intact virus may eliminate any possibility of discovering which particular human defence mechanism(s) the variola virus had evolved to evade and the understanding of the immune system we may gain from these studies. Identification of novel cytokine receptors or cytokines One of the most exciting prospects of studies on poxvirus pathogenesis is their potential to identify novel immune regulatory molecules. Several poxvirus genes encode proteins, either present on the cell surface or secreted from the cell, that contribute to virus virulence by an unknown mechanism and have no amino acid similarities to current databases (Fig. 2). These molecules may interact with as yet unidentified key immune regulatory molecules, thus representing viral homologues of novel cytokines or cytokine inhibitors. The best example to illustrate this is the vaccinia virus protein B18R. In common with the vaccinia IL-lPR, B18R has three immunoglobulin-like domains and sequence similarity to the cellular IL-lR, but no binding to IL-l has been observed. Surprisingly, the protein has recently been found to function as a soluble receptor for IFN-a/l3 (Ref. 26). The cellular receptors for IFN-o/l3 identified to date belong to a different family of proteins with

fibronectin type III domainP, thus B18R mav represent a homologue of an unidentified cellular II%-cu/PR that belongs to the immunoglobulin superfamily. Concluding remarks Viruses have survived under adverse host conditions during their evolution and have become adapted to block the action of key regulators of the immune and inflammatory responses. Studies on poxvirus mechanisms of immune evasion have identified virus proteins that block the activity of specific cytokines. These studies may unravel new physiological functions of cytokines and identify immune regulatory molecules that are not yet known. In addition, poxviruses provide a unique model to study the function of host cytokines in UZ’YO, which is often difficult to address and interpret in other models. Furthermore, the study of poxvirus pathogenesis constitutes a new and exciting field that may lead to the discovery of new strategies of immune modulation and new treatments for inflammatory diseases. We thank Begofia Aguado and Julian Symons for crirical reading of the manuscript. The work in the authors’ lahoratory was supported by The Wellcome Trust.

Antonio Alcami and Geoffrey Smith are at the Sir William Dunn School of Pathology, University of Oxford, Oxford, UK OX1 3RE. References 1 Fenner, F., Anderson, D.A., Arita, I., Jezek, Z. and Ladnyi, I.D. (1988) Smallpox and its Eradicatwn. World Health Organization 2 Fenner, F., Wittek, R. and Dumbell, K.R. (19891 The Oythopoxvi~uses, Academic Press 3 Moss, B. (1990) in Virology (Vol. 2) (Fields, B.N., Knipe, D.M., Chanock, R.M. et al., eds), pp. 2079-2 11I, Raven Press 4 Goebel, S.J., Johnson, G.P., Perkus, M.E., Davis, S.W., Winslow, J-P. and Paoletti, E. (1990) Virology 179, 247-266 5 Buller, R.M.L. and Palumbo, G.J. (1991) Micro&)/. Re~g. 55,80-122 6 Smith, G.L. (1993) I. Gen. Viyol. 74, 172.5-l 740 7 Smith, G.L. (1994) Trends Microbial. 2, 81-88 8 McFadden, G. and Graham, K. (1994) Semin. Viral. 5, 421-429 9 Spriggs, M.K. (1994) Curr. Opin. Immunol. 6, 526-529 10 McFadden, G. (1995) Viyoceptoys, Virokines and Related Immune Modulators Encoded by DNA Viruses, R.G. Landes Company

11 Smith, CA.. Davis. T., Wignall, J.M. etal. (1991) Biochem Bmphys. Res. Commun. 176, 335-342 12 Upton, C., Macen, J.L., Schreiber, M. and McFadden, G. (1991) Virology 1X4,370-382 13 Howard, S.T., Chdn, Y.S. and Smith, G.L. (1991) Virology 180, 633-647 14 Aguado, B.. Selmes, I.P. and Smith, G.L. ( 1992) /. Gen. Virol. 73. 2887-2902 15 Shchelkunov, S.N., Blinov, V.M. and Sandakhchiev, L.S. (1993) FEBS Lett. ,319, 80-83 16 Alcami, A. and Smith, G.L. (1993) FEBS Lett. 335, 136-137 17 Massung, R.F., Esposito, J.J., Liu. L-I. et al. (1993) Nature 366, 748-751 18 Massung, K.F., Llu, L-l., Qi, J. et al. (1994) Virology 201,215-240 19 Hu, F., Smith, CA. and Pickup, D.J. (1994) Virology 204,343-3.56 20 Spriggs, M.K., Hruby, D.E., Maliszewski, C.R. et al. (1992) Cell 71. 145-3.52 21 Alcami, A. and Smith. G.L. (1992) Cell 71, 153-167 22 Upton, C., Mossman, K. and McFadden, G. (1992) Science 258,1369-1372 23 Massung, R.F., Jayarama, V. and Moyer, R.W. (1993) Virology 197, 5 1l-528 24 Mossman, K., Upton, C., Buller, R.M.L. and McFadden, G. (1995) Vzrology 208, 762-769 25 Alcami, A. and Smith, G.L. (1995) J. Vzrol. 69,4633-4639 26 Symons, J.A., Alcami. A. and Smith, G.I.. (1995) Cell 81, 551-560 27 Ahuja, S.K., Gao, J-L. and Murphy, P.M. (19941 lmmunol. Today 15,281-287 28 Hsu, D-H., de Waal Malefyt, R., Fiorentino, D.F. et al. (I 990) Science 250, 830-832 29 Wold, W.S.M., Hermiston, T.W. and Tollefson, 4.E. (1994) Trends Microbtol. 2,437-443 30 Dinarello, CA. (1994) FASEB 1. 8, 1314-1325 31 Kluger, M.J. (1991) Physzoi. Rev. 71, 93-127 32 Ramshaw, I., Ruby, J., Ramsay, A., Ada, G. and Karupiah, G. (1992) Immunol. Rev. 127, 157-182 33 Colotta, F., Dower, S.K., Sims, J.E. and Mantovani, A. (1994) lmmunol. Today 15,562-566 34 Rose-John, S. and Heinrich, P.C. (1994) Biochem. /. 300, 281-290 35 Schreiber, M. and McFadden, G. (1994) Virology 204, 692-705 36 Mossman, K., Upton, C. and McFadden, G. (199.5) 1. Biol. Chem. 270, 3031-3038 37 Dinarello, CA. (1993) Immunol. Today 14,260-264 38 Callard, R. and Gearmg, A. (1994) The Cytokine FactsBook, Academic Press 39 Duncan, S.A. and Smith, G.L. (1992) J. Viral. 66, 1610-1621

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