Smallpox inhibitor of complement enzymes (SPICE): dissecting functional sites and abrogating activity.
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 approximately 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.
Liszewski
M Kathryn
MK
Department of Medicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Leung
Marilyn K
MK
Hauhart
Richard
R
Fang
Celia J
CJ
Bertram
Paula
P
Atkinson
John P
JP
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001666:Binding Sites, Antibody; D016466:CHO Cells; D003166:Complement Activating Enzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006025:Glycosaminoglycans / Q000037:antagonists & inhibitors / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D012901:Variola virus / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D037521:Virulence Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology
D000911:Antibodies, Monoclonal; D006025:Glycosaminoglycans; C532808:SPICE protein, variola virus; D014763:Viral Matrix Proteins; D037521:Virulence Factors; D003179:Complement C3b; D006493:Heparin; D003166:Complement Activating Enzymes