Influence of glycosylation and oligomerization of vaccinia virus complement control protein on level and pattern of functional activity and immunogenicity

Abstract

Vaccinia virus complement control protein (VCP) is one of the proteins encoded by vaccinia virus to modulate the host inflammatory response. VCP modulates the inflammatory response and protects viral habitat by inhibiting the classical and the alternative pathways of complement activation. The extended structure of VCP, mobility between its sequential domains, charge distribution and type of residues at the binding regions are factors that have been identified to influence its ability to bind to complement proteins. We report that a Lister strain of vaccinia virus encodes a VCP homolog (Lis VCP) that is functional, glycosylated, has two amino acids less than the well-characterized VCP from vaccinia virus WR strain (WR VCP), and the human smallpox inhibitor of complement enzymes (SPICE) from variola virus. The glycosylated VCP of Lister is immunogenic in contrast to the weak immunogenicity of the nonglycosylated VCP. Lis VCP is the only orthopoxviral VCP homolog found to be glycosylated, and we speculate that glycosylation influences its pattern of complement inhibition. We also correlate dimerization of VCP observed only in mammalian and baculovirus expression systems to higher levels of activity than monomers, observed in the yeast expression system.

Keywords

vaccinia virus / complement / glycosylation / oligomerization / structure-function / immunogenicity

References

[1] Adamo, J.E., Meseda, C.A., Weir, J.P., and Merchlinsky, M.J. (2009). Smallpox vaccines induce antibodies to the immunomodulatory, secreted vaccinia virus complement control protein. J Gen Virol 90, 2604–2608 .10.1099/vir.0.008474-019587131
[2] Al-Mohanna, F., Parhar, R., and Kotwal, G.J. (2001). Vaccinia virus complement control protein is capable of protecting xenoendothelial cells from antibody binding and killing by human complement and cytotoxic cells. Transplantation 71, 796–801 .10.1097/00007890-200103270-0001911330545
[3] Anderson, J.B., Smith, S.A., van Wijk, R., Chien, S., and Kotwal, G.J. (2003). Vaccinia virus complement control protein inhibits hyperacute xenorejection in a guinea pig-to-rat heterotopic cervical cardiac xenograft model by blocking both xenoantibody binding and complement pathway activation. Transpl Immunol 11, 129–135 .10.1016/S0966-3274(02)00149-112799195
[4] Bender, B.S., Rowe, C.A., Taylor, S.F., Wyatt, L.S., Moss, B., and Small, P.A. Jr. (1996). Oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza. J Virol 70, 6418–6424 .8709274
[5] Cieslak, T.J., Christopher, G.W., Kortepeter, M.G., Rowe, J.R., Pavlin, J.A., Culpepper, R.C., and Eitzen, E.M. Jr. (2000). Immunization against potential biological warfare agents. Clin Infect Dis 30, 843–850 .10.1086/31381210880299
[6] Dowling, W., Thompson, E., Badger, C., Mellquist, J.L., Garrison, A.R., Smith, J.M., Paragas, J., Hogan, R.J., and Schmaljohn, C. (2007). Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines. J Virol 81, 1821–1837 .10.1128/JVI.02098-0617151111
[7] Empig, C., Kenner, J.R., Perret-Gentil, M., Youree, B.E., Bell, E., Chen, A., Gurwith, M., Higgins, K., Lock, M., Rice, A.D., (2006). Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge. Vaccine 24, 3686–3694 .10.1016/j.vaccine.2005.03.02916430997
[8] Fenner, F., Henderson, D.A., Arita, I., Jezek, Z., and Ladnyi, I.D. (1988) Smallpox and its eradication. Geneva: World Health Organization
[9] Ganesh, V.K., Smith, S.A., Kotwal, G.J., and Murthy, K.H.M. (2004). Structure of vaccinia complement protein in complex with heparin and potential implications for complement regulation. Proc Natl Acad Sci USA 101, 8924–8929 .10.1073/pnas.040074410115178763
[10] Garcel, A., Crance, J.-M., Drillien, R., Garin, D., and Favier, A.-L. (2007). Genomic sequence of a clonal isolate of the vaccinia virus Lister strain employed for smallpox vaccination in France and its comparison to other orthopoxviruses. J Gen Virol 88, 1906–1916 10.1099/vir.0.82708-017554021
[11] Garcel, A., Perino, J., Crance, J.M., Drillien, R., Garin, D., and Favier, A.L. (2009). Phenotypic and genetic diversity of the traditional Lister smallpox vaccine. Vaccine 27, 708–717 .10.1016/j.vaccine.2008.11.06319059294
[12] Ghebremariam, Y.T. (2006). Ph. D. thesis. University of Cape Town, South Africa
[13] Ghebremariam, Y.T., Odunuga, O.O., Janse, K., and Kotwal, G.J. (2005). Humanized recombinant vaccinia virus complement control protein (hrVCP) with three amino acid changes, H98Y, E102K, and E120K creating an additional putative heparin binding site, is 100-fold more active than rVCP in blocking both classical and alternative complement pathways. Ann N Y Acad Sci 1056, 113–122 .10.1196/annals.1352.02416387681
[14] Henderson, D.A., Inglesby, T.V., Bartlett, J.G., Ascher, M.S, Eitzen, E., Jahrling, P.B., et al (1999). Smallpox as a Biological Weapon. JAMA 281, 2127–2137 .
[15] Ichinose, A., Bottenus, R.E., and Davie, E.W. (1990). Structure of transglutaminases. J Biol Chem 265, 13411–13414 .1974250
[16] Isaacs, S.N., Argyropoulos, E., Sfyroera, G., Mohammad, S., and Lambris, J.D. (2003). Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein. J Virol 77, 8256–8262 .10.1128/JVI.77.15.8256-8262.200312857894
[17] Jha, P., Smith, S.A., Justus, D.E., and Kotwal, G.J. (2005). Vaccinia virus complement control protein ameliorates collagen-induced arthritic mice. Ann N Y Acad Sci 1056, 55–68 .10.1196/annals.1352.00416387677
[18] Joklik, W.K. (1962). The purification fo four strains of poxvirus. Virology 18, 9–18 .10.1016/0042-6822(62)90172-114036977
[19] Kahn, D., Smith, S.A., and Kotwal, G.J. (2003). Dose-dependent inhibition of complement in baboons by vaccinia virus complement control protein: implications in xenotransplantation. Transplant Proc 35, 1606–1608 .10.1016/S0041-1345(03)00485-812826233
[20] Kang, Y.H., Tan, L.A., Carroll, M.V., Gentle, M.E., and Sim, R.B. (2009). Target pattern recognition by complement proteins of the classical and alternative pathways. Adv Exp Med Biol 653, 117–128 .10.1007/978-1-4419-0901-5_819799115
[21] Kotwal, G.J. (1994). Purification of virokines using ultrafiltration. Am Biotechnol Lab 12, 76–77 .7765357
[22] Kotwal, G.J. (2000). Poxviral mimicry of complement and chemokine system components: what’s the end game? Immunol Today 21, 242–248 .10.1016/S0167-5699(00)01606-610782056
[23] Kotwal, G.J., and Abrahams, M.R. (2004). Growing poxviruses and determining virus titer. Methods Mol Biol 269, 101–112 .15114010
[24] Kotwal, G.J., Isaacs, S.N., McKenzie, R., Frank, M.M., and Moss, B. (1990). Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 250, 827–830. 10.1126/science.22374342237434
[25] Kotwal, G.J., Miller, C.G., and Justus, D.E. (1998a). The inflammation modulatory protein (IMP) of cowpox virus drastically diminishes the tissue damage by down-regulating cellular infiltration resulting from complement activation. Mol Cell Biochem 185, 39–46. 10.1023/A:10068446248259746210
[26] Kotwal, G.J., and Moss, B. (1988). Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins. Nature 335, 176–178 .10.1038/335176a03412473
[27] Kotwal, G.J., Reynolds, D., Keeling, K., Howard, J., and Justus, D.E. (1998b) Vaccinia virus complement control protein is a virokine with lysozyme-like heparin-binding activity: possible implications in prolonged evasion of host immune response. In: Proceedings of the 10th International Congress of Immunology . Talwar, G. P., Nath, I., Ganguly, N. K., and Rao, K. V. S. (ed.), New Delhi, 315–320 .
[28] Liszewski, M.K., and Atkinson, J.P. (1996). Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement. J Immunol 156, 4415–4421 .8666815
[29] Liszewski, M.K., Leung, M., Cui, W., Subramanian, V.B., Parkinson, J., Barlow, P.N., Manchester, M., and Atkinson, J.P. (2000). Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46). J Biol Chem 275, 37692–37701 .10.1074/jbc.M00465020010960475
[30] Liszewski, M.K., Leung, M.K., and Atkinson, J.P. (1998). Membrane cofactor protein: importance of N- and O-glycosylation for complement regulatory function. J Immunol 161, 3711–3718 .9759896
[31] Liszewski, M.K., Leung, M.K., Hauhart, R., Buller, R.M., Bertram, P., Wang, X., Rosengard, A.M., Kotwal, G.J., and Atkinson, J.P. (2006). Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation. J Immunol 176, 3725–3734 .16517741
[32] McKenzie, R., Kotwal, G.J., Moss, B., Hammer, C.H., and Frank, M.M. (1992). Regulation of complement activity by vaccinia virus complement-control protein. J Infect Dis 166, 1245–1250 .1431243
[33] Miller, C.G., Justus, D.E., Jayaraman, S., and Kotwal, G.J. (1995). Severe and prolonged inflammatory response to localized cowpox virus infection in footpads of C5-deficient mice: investigation of the role of host complement in poxvirus pathogenesis. Cell Immunol 162, 326–332 .10.1006/cimm.1995.10867743560
[34] Miller, C.G., Shchelkunov, S.N., and Kotwal, G.J. (1997). The cowpox virus encoded homolog of vaccinia virus is an inflammation modulatory protein. Virology 33, 126–133 .912385410.1006/viro.1996.8396
[35] Morikawa, S., Sakiyama, T., Hasegawa, H., Saijo, M., Maeda, A., Kurane, I., Maeno, G., Kimura, J., Hirama, C., Yoshida, T., (2005). An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection. J Virol 79, 11873–11891 .10.1128/JVI.79.18.11873-11891.200516140764
[36] Morley, B.J., Campbell, R.D.(1984). Internal homologies of the Ba fragment from human complement component Factor B, a class III MHC antigen. EMBO J 3,153–157 .6323161
[37] Murthy, K.H.M., Smith, S.A., Ganesh, V.K., Judge, K.W., Mullin, N., Barlow, P.N., Ogata, C.M., and Kotwal, G.J. (2001). Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans. Cell 104, 301–311 .10.1016/S0092-8674(01)00214-811207370
[38] Norman, D.G., Barlow, P.N., Baron, M., Day, A.J., Sim, R.B., Campbell, I.D.(1991). Three-dimensional structure of a complement control protein module in solution. J Mol Biol 219, 717– 725 .1829116
[39] Ober, B.T., Brühl, P., Schmidt, M., Wieser, V., Gritschenberger, W., Coulibaly, S., Savidis-Dacho, H., Gerencer, M., and Falkner, F.G. (2002). Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara. J Virol 76, 7713–7723 10.1128/JVI.76.15.7713-7723.2002.12097585
[40] Odunuga, O.O., Ghebremariam, Y.T., and Kotwal, G.J. (2005). Lister homologue of vaccinia virus complement control protein is two amino acids shorter, has putative glycosylation sites and other functional and structural differences. FEBS J 272(Supplement 1), 15–16 .
[41] Reynolds, D.N., Keeling, K.L., Molestina, R., Srisatjaluk, R., Butterfield, J.H., Ehringer, W., (2000) Heparin binding activity of vaccinia virus complement control protein confers additional properties of uptake by mast cells and attachment to endothelial cells. In: Advances in Animal Virology , Jameel, S., and Lambris, J. D., ed. New York: Oxford & IBH, 237–342 .
[42] Rosengard, A.M., Liu, Y., Nie, Z., and Jimenez, R. (2002). Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc Natl Acad Sci U S A 99, 8808–8813 .12034872
[43] Rosenthal, S.R., Merchlinsky, M., Kleppinger, C., and Goldenthal, K.L. (2001). Developing new smallpox vaccines. Emerg Infect Dis 7, 920–926 .10.3201/eid0706.01060211747717
[44] Sahu, A., Isaacs, S.N., Soulika, A.M., and Lambris, J.D. (1998). Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway. J Immunol 160, 5596–5604 .9605165
[45] Scott, M.J., Burch, P.T., Jha, P., Peyton, J.C., Kotwal, G.J., and Cheadle, W.G. (2003). Vaccinia virus complement control protein increases early bacterial clearance during experimental peritonitis. Surg Infect (Larchmt) 4, 317–326 .10.1089/10962960332276137315012858
[46] Sfyroera, G., Katragadda, M., Morikis, D., Isaacs, S.N., and Lambris, J.D. (2005). Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins. J Immunol 174, 2143–2151 .15699145
[47] Smith, S.A., and Kotwal, G.J. (2001). Virokines: novel immunomodulatory agents. Expert Opin Biol Ther 1, 343–357 .10.1517/14712598.1.3.34311727510
[48] Smith, S.A., Krishnasamy, G., Murthy, K.H.M., Cooper, A., Bromek, K., Barlow, P.N., and Kotwal, G.J. (2002). Vaccinia virus complement control protein is monomeric, and retains structural and functional integrity after exposure to adverse conditions. Biochim Biophys Acta 1598, 55–64 .12147344
[49] Smith, S.A., Mullin, N.P., Parkinson, J., Shchelkunov, S.N., Totmenin, A.V., Loparev, V.N., Srisatjaluk, R., Reynolds, D.N., Keeling, K.L., Justus, D.E., (2000). Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense. J Virol 74, 5659–5666 .10.1128/JVI.74.12.5659-5666.200010823874
[50] Stannard, L.M., Marais, D., Kow, D., and Dumbell, K.R. (1998). Evidence for incomplete replication of a penguin poxvirus in cells of mammalian origin. J Gen Virol 79, 1637–1646 .9680125
[51] Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680 .10.1093/nar/22.22.46737984417
[52] Wittek, R. (2006). Vaccinia immune globulin: current policies, preparedness, and product safety and efficacy. Int J Infect Dis 10, 193–201 .10.1016/j.ijid.2005.12.00116564720