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Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity¹

M. Kathryn Liszewski^*, Marilyn K. Leung^*, Richard Hauhart^*, Celia J. Fang, Paula Bertram^* and John P. Atkinson^2,*

^* Department of Medicine/Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110; and Medical Service, Veterans Administration Medical Center, San Francisco, CA 94121

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research.

² Address correspondence and reprint requests to Dr. John P. Atkinson, Department of Medicine/Division of Rheumatology, Washington University School of Medicine, Campus Box 8045, 660 South Euclid, St. Louis, MO 63110. E-mail address: jatkinso{at}dom.wustl.edu

³ Abbreviations used in this paper: PICES, poxviral inhibitors of complement enzymes; CA, cofactor activity; CCP, complement control protein; CHO, Chinese hamster ovary (cells); DAA, decay-accelerating activity; DAF, decay-accelerating factor; GAG, glycosaminoglycan; MCP, membrane cofactor protein (CD46); MOPICE, monkeypox inhibitor of complement enzymes; SPICE, smallpox inhibitor of complement enzymes; VICE, vaccinia inhibitor of complement enzymes; VIG, Vaccinia Immune Globulin; VCP, vaccinia complement control protein.