Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement

Abstract

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

Figure 1

Sequences of SPICE and VCP; identification of rSPICE, rVCP, wild-type VCP. (A) Aligned amino acid sequences of SPICE and VCP, with arrows identifying residue differences. Conserved cysteines that dictate SCR folding are schematically linked, and the last amino acid of each SCR is numbered. (B) Front and back schematic views of SPICE mapped onto the structure of VCP, visualized by RASMOL V.2.7.2.1. The amino acid differences are pink in SCR2, orange in SCR3, and green in SCR4. (C) Coomassie-stained SDS/PAGE of SPICEhis, VCPhis, SPICEFc, and VCPFc. (D) Western blot of VCPhis, SPICEhis, and wild-type VCP. Proteins were identified by using polyclonal anti-VCP/SPICE antiserum. Wild-type VCP and VCPhis migrate identically at approximately 35 kDa.

Figure 2

C3b and C4b degradation in the presence of SPICEhis and VCPhis. (A) Appearance of C3b cleavage products in the presence of factor I and either factor H, VCPhis, SPICEhis, sCR1, or PBS at 0, 4, and 24 h. (B) Schematic of C3b degradation in the presence of factor I (45) and cofactors, VCP or SPICE. (C) Appearance of C4b cleavage products in the presence of factor I and either factor H, VCPhis, SPICEhis, sCR1, or PBS at 0, 4, and 24 h. (D) Schematic of C4b degradation (46) in the presence of factor I and cofactors, VCP or SPICE.

Figure 3

Time course of C3b degradation with SPICEhis, VCPhis, and sCR1. (A) Coomassie-stained SDS/PAGE of C3b degradation products from reactions containing C3b, factor I, and either SPICEhis, VCPhis, or sCR1. Reaction samples were removed at 0, 5, 15, 30, 60, and 120 min. (B) Rate of degradation of C3bα′. Gels were scanned for densitometric analysis by using transmissive spectrophotometer, and the OD of the C3bα′ band at each time point was compared with the OD of the original sample of C3bα′. The percent degradation is plotted against time. Experiments were performed five times and error bars represent SD. (C) Rate of degradation of C3bα′ chain and the generation of other C3b cleavage products in the presence of SPICEhis are plotted as a percent of the original C3bα′ chain. Analysis was performed as described in B. (D) Rate of degradation of C3b and generation of C3b cleavage products in the presence of VCPhis are plotted as a percentage of the original C3bα′ chain. Analysis was performed as described in B.

Figure 4

Time course of C4b degradation with SPICEhis, VCPhis, and sCR1. (A) Coomassie-stained SDS/PAGE of C4b degradation products from reactions containing C4b, factor I, and either SPICEhis, VCPhis, or sCR1. Reaction samples were removed at 0, 30, 60, 120, 180, and 240 min for SPICEhis and VCPhis and 0, 5, 15, 30, 60, and 120 min for sCR1. (B) Rate of degradation of C4bα′. The gels were scanned for densitometric analysis by using transmissive spectrophotometer and the OD of the C4bα′ band at each time point was compared with the OD of the original sample of C4bα′. The percent degradation is plotted against time. Experiments were performed five times and error bars represent SD. (C) Rate of degradation of C4bα′ chain and generation of C4b cleavage products in the presence of SPICEhis are plotted as a percent of the original C4bα′ chain. Analysis was performed as described in B. (D) Rate of degradation of C4b and generation of C4b cleavage products in the presence of VCPhis are plotted as a percentage of the original C4bα′ chain. Analysis was performed as described in B.

Figure 5

Inhibition of complement from different species by viral CRPs. (A) Influence of viral CRPs on the classical pathway-mediated hemolysis of EA in the presence of human complement. EA were incubated with human serum in the presence of no inhibitor (black squares), or equimolar amounts of SPICEhis (red diamonds) and VCPhis (green circles) or SPICEFc (blue triangles) and VCPFc (aqua quartered squares) for 1 h at 37°C. Reported values represent the amount of hemolysis relative to maximum hemolysis, as determined by the addition of water to EA. Results are representative of ten separate experiments. (B) Influence of viral CRPs on the classical pathway-mediated hemolysis of EA in the presence of dog complement. Experiments were performed as described above except that dog serum was used as the source of complement. Results are representative of six separate experiments. (C) Influence of viral CRPs on the alternative pathway-mediated hemolysis of Er in the presence of human complement. Results are representative of four separate experiments. (D) Influence of viral CRPs on the alternative pathway-mediated hemolysis of Er in the presence of guinea pig complement. Results are representative of three separate experiments.