Identification of Complement Regulatory Domains ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 2005, p. 12382–12393 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.19.12382–12393.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 79, No. 19

Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein Jayati Mullick,† John Bernet,† Yogesh Panse, Sharanabasava Hallihosur, Akhilesh K. Singh, and Arvind Sahu* National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India Received 22 March 2005/Accepted 7 July 2005

Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP’s various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.

The complement system is an ancient and one of the principal innate immune defense mechanisms against all invading pathogens, including viruses (7, 34). It has a unique ability to recognize and label pathogens as non-self, with and without the help of pattern recognition molecules (7, 27). Apart from its direct effect on pathogens, it also plays an additional role in initiation and enhancement of the pathogen-specific adaptive immune responses (6, 34, 42). A significant body of knowledge indicates that both acute and latent viruses are susceptible to neutralization by the complement system (3, 7, 25). Thus, complement exerts a strong selective pressure on viruses during infection. To survive as successful pathogens, viruses have evolved different strategies to subvert the complement system. Efficient complement evasion mechanisms evolved by viruses include direct complement inactivation by encoding structural and/or functional homologs of host complement regulatory proteins or by capturing host membrane regulatory proteins and using host receptors for cellular entry (3, 25). Important examples include viruses belonging to the family of poxviruses, herpesviruses, retroviruses, paramyxoviruses, and picornaviruses (1, 10, 28, 30, 33, 51, 56). Vaccinia virus (VV), a prototype of the family Poxviridae and genus Orthopoxvirus, is a cytoplasmic large DNA virus (18, 32). VV embodies an illustrious example of complement evasion. It is known to have developed two efficient mechanisms to evade the complement system. (i) It encodes within its genome a

* Corresponding author. Mailing address: National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India. Phone: 91-20-2569-0922. Fax: 91-20-2569-2259. E-mail: arvindsahu @nccs.res.in. † J.M. and J.B. contributed equally to this work.

27-kDa protein, homologous to human complement regulators, known as VV complement control protein (VCP), which is a potent soluble complement inhibitor (22, 29, 46). (ii) It incorporates host complement proteins (membrane cofactor protein [MCP], CD46; decay-accelerating factor [DAF], CD55; and CD59) while budding, rendering itself resistant to complement attack (57). VCP, encoded by the C21L gene, is one of the first viral proteins identified to have complement-binding activity (23). The primary structure of VCP consists of 244 amino acids with a signal peptide of 19 amino acids. It comprises four CCP domains that are typically present in human regulators of complement activation (RCA) proteins and exhibits considerable sequence similarity to factor H (26%), C4b-binding protein (37%), MCP (35%), DAF (38%), and complement receptor 1 (CR1) (37%). The nuclear magnetic resonance structure of VCP modules (CCPs 2 to 3 and CCPs 3 to 4) revealed that each CCP folds into a compact six-␤-strand structure similar to CCP 16 of factor H (13). Recent elucidation of the crystal structure of VCP depicted it as a highly extended molecule with heparinbinding site at its C-terminal region (9, 36). An interesting feature revealed by the crystal structure was the charge distribution of VCP domains: CCP domains 1 and 4 are surrounded by a positive field, while the middle two domains (CCPs 2 and 3) are surrounded by a predominantly negative field. Functional studies revealed that VCP protects vaccinia virions from antibody-dependent complement-mediated neutralization (15); vaccinia virus mutants that do not express VCP are attenuated in vivo (15). Initial studies of the mechanism of complement inhibition performed using both partially purified VCP and culture medium containing secreted VCP have shown that it inhibits the classical pathway-mediated lysis of

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sheep erythrocytes, binds to C3b and C4b, and accelerates the decay of the classical and (to a limited extent) the alternative pathway C3 convertases (29). A later study performed using purified recombinant VCP demonstrated that it possesses factor I cofactor activity for C3b and C4b and is a poor inhibitor of the alternative pathway, in comparison to the classical pathway (46). Recently, the binding mechanism of VCP with C3b and C4b has been characterized using surface plasmon resonance (SPR). The data suggested that binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions, as is observed for factor H and CR1 (2). Further, binding to C3b and C4b involves ionic interactions, and the binding site of VCP on C3b and C4b is located within the C3dg and C4c regions, respectively (2). Although significant efforts were made to establish VCP as a complement inhibitor (3, 17), no efforts were made to shed light on the identification of its domains important for its complement regulatory activities: factor I cofactor activity for C3b and C4b, and decay-accelerating activity for the classical and alternative pathway C3 convertases. In this paper, we have identified which of the four CCPs of VCP are required to confer cofactor activities, decay-accelerating activities, and binding to target proteins C3b and C4b. Our results suggest that a minimum of three CCP domains are required for its cofactor activities, binding to C3b and C4b, and its limited alternative pathway decay-accelerating activity and that two domains are enough to mediate the classical pathway decayaccelerating activity. MATERIALS AND METHODS Reagents and buffers. Antibody-coated sheep erythrocytes (designated EA) were prepared by incubating the sheep erythrocytes with anti-sheep erythrocyte antibodies (ICN Biomedical, Inc., Irvine, Calif.). Antiserum recognizing VCP was raised in rabbit by immunizing with purified VCP. Buffers used were VBS (5 mM barbital and 145 mM NaCl, pH 7.4), GVB (VBS containing 0.1% gelatin), DGVB (2.5 mM barbital and 73 mM NaCl, 0.1% gelatin, and 2.5% dextrose), DGVB⫹⫹ (DGVB containing 0.5 mM MgCl2 and 0.15 mM CaCl2), and phosphate-buffered saline (PBS) (10 mM sodium phosphate and 145 mM NaCl, pH 7.4). Mg EGTA contained 0.1 M MgCl2 and 0.1 M EGTA. Purified complement proteins. The human complement factors B, D, H, and I were kindly provided by Michael K. Pangburn (University of Texas Health Center, Tyler, Tex.) and the recombinant human soluble CR1 (sCR1) was a generous gift of Henry Marsh (AVANT Immunotherapeutics, Inc., Needham, Mass.). Human C1, C4, and C2 were purchased from Calbiochem (La Jolla, Calif.). Human C3 was purified according to Hammer et al. (12) with minor modifications. Twenty parts of human plasma were mixed with 1 part of inhibitor solution (1 M KH2PO4, 0.2 M Na4 EDTA, 0.2 M benzamidine, and 1 mM phenylmethylsulfonyl fluoride) and precipitated first with 4.5% polyethylene glycol and then with 12% polyethylene glycol at 0°C. The 12% pellet was then dissolved in 20 mM sodium phosphate, pH 7.4, and loaded onto a Source Q column (1.2 by 9.5 cm; Amersham Pharmacia Biotech, Uppsala, Sweden). The bound proteins were eluted with a linear gradient of 0 to 0.5 M NaCl. Fractions containing C3 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunodiffusion, pooled, and loaded onto a Mono Q 10/10 column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate, pH 7.9. Bound proteins were eluted with a linear salt gradient of 0 to 0.5 M and subjected to SDS-PAGE analysis. C3-containing fractions were pooled, concentrated by ultrafiltration, and loaded onto Superose 12 (two columns connected in a series; Amersham Pharmacia Biotech) in PBS. Homogeneous C3 fractions as analyzed by SDS-PAGE were pooled. The purified C3 contained ⬎90% native C3, as determined by analyzing the protein sample on a Mono S column (Amersham Pharmacia Biotech) (39). C3b was generated by limited trypsin cleavage and purified on a Mono Q 5/5 column (46). Human C4b used was purified as previously described (2) or purchased from Calbiochem (La Jolla, Calif.). Construction of deletion mutants. The deletion constructs comprising CCPs 1 to 3, 2 to 4, 1 to 2, 2 to 3, and 3 to 4 of VCP were constructed from full-length

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VCP (46) by PCR amplification and cloned into the yeast expression vector pPICZ␣ (Invitrogen, Carlsbad, Calif.), downstream of the AOX1 methanolinducible promoter by the standard protocol. For amplification of the respective VCP deletion mutants, the following sequence-specific primers were used: for CCP 1-3, AS-1 (5⬘-GGAATTCTGCTGTACTATTCCGTCACGACCC-3⬘) and AS-5 (5⬘-GCTCTAGATTATTTAACAATCTGACACGTGGGTGG-3⬘); for CCP 2-4, AS-2 (5⬘-GGAATTCTGCCCATCGCCTCGAGATATCG-3⬘) and AS-4 (5⬘-GCTCTAGATTAGCGTACACATTTTGGAAGTTCCG-3⬘); for CCP 1-2, AS-1 and AS-6 (5⬘-GCTCTAGATTATTTAACAGATTCACAAATAGGT GCC-3⬘); for CCP 2-3, AS-2 and AS-5; and for CCP 3-4, AS-3 (5⬘-GGAATTC TGCCAATCCCCTCCATCTATATCCAACGG-3⬘) and AS-4. The primers incorporated restriction sites EcoRI and XbaI (underlined) in the forward and the reverse orientations, respectively, and a stop codon (in boldface type) in the reverse orientation primers. The amplification was carried out using Titanium Taq DNA polymerase (B. D. Biosciences Clonetech, Palo Alto, CA), with an initial cycle of denaturation at 94°C for 2 min, followed by 25 cycles of 1 min at 94°C, 1 min at 60°C or 65°C according to the annealing temperature of the primer, 1 min at 72°C, and a final extension for 10 min at 72°C. All the PCR products were digested with EcoRI and XbaI and cloned into pPICZ␣ EcoRI and XbaI cut vector. The validity of all the constructs was confirmed by automated DNA sequencing. Five micrograms of each of the SacI-digested pPICZ␣ VCP deletion constructs was then integrated into Pichia pastoris as per the manufacturer’s protocol. The integration of the deletion mutants in Pichia were confirmed by amplifying the respective genomic DNA using the 5⬘AOX1 and 3⬘AOX1 primers and with a combination of gene-specific primers. Further, the validated integrants were used for expression of the recombinant proteins. Expression and purification of vaccinia virus complement control protein and its deletion mutants. The vaccinia virus complement control protein cloned in Pichia pastoris was expressed and purified as previously described (2). Expression of VCP mutants was performed as described below. A single colony of recombinant Pichia pastoris expressing the respective VCP mutant was inoculated in 10 ml of BMGY medium (100 mM potassium phosphate, pH 6.0, 10 g/liter yeast extract, 20 g/liter peptone, 13.4 g/liter yeast nitrogen base, 0.4 mg/liter biotin, and 1% glycerol) and incubated overnight at 30°C in a shaking incubator. This inoculum was then added to 400 ml of BMGY and incubated for 48 h at 30°C with shaking. The cells were centrifuged, resuspended in 400 ml of BMMY (BMGY containing 0.5% methanol, but without 1% glycerol), and incubated at 30°C for an additional 96 h with vigorous shaking; 0.5% methanol was added after every 24 h. After incubation, cells were pelleted and the supernatant containing the VCP mutant was collected for purification. For purification, the supernatants containing VCP mutants were concentrated by ultrafiltration and precipitated with 80% ammonium sulfate at 0°C. The pellets were then suspended and dialyzed in PBS. For further purification, CCP 1-3 and CCP 1-2 mutants were passed through a PD-10 column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate (pH 7.4), loaded onto a heparin-agarose column in the same buffer, and eluted with 500 mM NaCl. The eluted proteins were then exchanged into 20 mM Tris (pH 7.9), loaded onto a Mono Q column, and eluted with a linear gradient of 0 to 500 mM NaCl. For purification of CCP 2-4 and CCP 2-3 mutants, the samples were passed through DEAE-Sephacel (Sigma, St. Louis, Mo.) preequilibrated with 10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl. The flowthrough was collected, passed through a PD-10 column in 20 mM Tris (pH 7.9), and loaded onto a Mono Q column. The bound proteins were eluted with a linear gradient of 0 to 500 mM NaCl. CCP 3-4 mutant was purified by being loaded onto DEAESephacel in 10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl. The flowthrough containing CCP 3-4 mutant was collected and buffer exchange was performed using a PD-10 column preequilibrated with 5 mM sodium acetate, pH 4.0. The sample was then loaded onto a Mono S column, and the bound proteins were eluted with a linear salt gradient of 0 to 500 mM NaCl. Eluted fractions in all the above purifications were analyzed by SDS-PAGE and Western blotting using anti-VCP antibodies; fractions containing pure proteins were pooled, dialyzed into PBS, and concentrated by ultrafiltration. Mass analysis and protein sequencing. The molecular mass determination and sequencing of the VCP mutants were performed at the Biomolecular Research Facility (University of Virginia, Charlottesville, VA) as described below. The molecular weights of the VCP mutants were determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MS) using an Applied Biosystems Voyager DE-Pro. The samples were dissolved in 70% acetonitrile– 30% water, mixed with sinapinic acid matrix, and spotted on the matrix-assisted laser desorption ionization plate. Approximately 200 spectra were averaged for each sample. The sequencing of VCP mutants was performed as described below. The samples were run on a 12% SDS-PAGE gel and stained with Coomassie blue.

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The protein bands were cut, transferred to a siliconized tube, washed, and destained overnight in 200 ␮l of 50% methanol. The gel pieces were then dehydrated in acetonitrile, rehydrated in 30 ␮l of 10 mM dithiolthreitol (DTT) in 0.1 M ammonium bicarbonate, and reduced at room temperature for 30 min. The DTT solution was then removed, and the samples were alkylated in 30 ␮l of 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 30 min. The reagent was removed, and the gel pieces were dehydrated in 100 ␮l of acetonitrile. The gel pieces were subjected to two cycles of dehydration and rehydration in acetonitrile and 0.1 M ammonium bicarbonate and then dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/␮l trypsin (second band for each sample in chymotrypsin) in 50 mM ammonium bicarbonate on ice for 10 min, excess enzyme solution was removed, and 20 ␮l of 50 mM ammonium bicarbonate was added. The samples were digested overnight at 37°C, and the peptides formed were extracted from the polyacrylamide in two 30-␮l aliquots of 50% acetonitrile–5% formic acid. These extracts were combined and evaporated to 25 ␮l for MS analysis, which was done using a liquid chromatography-MS system consisting of a Finnigan LCQ ion trap mass spectrometer with a Protana nanospray ion source interfaced to a self-packed 8 cm by 75 ␮m (inner diameter) Phenomenex Jupiter 10-␮m C18 reversed-phase capillary column. The digest was analyzed using the double-play capability of the instrument acquiring one full scan mass spectrum to determine peptide molecular weights, followed by four product ion spectra to determine amino acid sequences in sequential scans. The data were analyzed by database searching using the Sequest algorithm and by manual interpretation. SPR measurements. The kinetics of binding of VCP and VCP mutants to human C3b and C4b were determined by using the SPR-based biosensor BIACORE 2000 (Biacore AB, Uppsala, Sweden). Binding experiments were performed using physiologic ionic strength buffer (PBS-T, 10 mM sodium phosphate, 145 mM NaCl, pH 7.4, containing 0.05% Tween-20) at 25°C. Addition of 0.05% Tween-20 blocked the nonspecific adsorption of VCP to the sensor chips (50). In this assay, both C3b and C4b were oriented in their physiological orientation on a streptavidin chip (Sensor Chip SA, Biacore AB) by labeling their free -SH group with biotin (2). In brief, 914 response units (RUs) of C3b and 916 RUs of C4b were immobilized on FC-2 and FC-3, respectively, and FC-1 served as a control flow cell. Binding was measured at 50 ␮l/min to avoid the mass transport effect, and it was measured for 120 s. Dissociation was measured for 180 s. The sensor chip surfaces were regenerated with 30-s pulses of 0.2 M sodium carbonate, pH 9.5. Sensograms obtained for the control flow cell (Fc-1) were subtracted from the data for the flow cell immobilized with either C3b or C4b, and the SPR data were analyzed by BIAevaluation software version 4.1 using global fitting. Measurement of factor I cofactor activity. Both qualitative and quantitative analyses of factor I cofactor activity of VCP and its mutants for C3b and C4b were determined using 10 mM phosphate buffer, pH 7.4, containing 145 mM NaCl. In these assays, 2.7 ␮g of C3b or 2.9 ␮g of C4b was mixed with 100 ng of factor I and VCP (as indicated in the figure legends) or its mutants and incubated at 37°C for 4 h in a total volume of 15 ␮l. The reactions were stopped by the addition of sample buffer containing DTT and electrophoresed on a 9% SDSPAGE gel for determining C3b cleavages and a 10% SDS-PAGE for C4b cleavages. The cleavage products were visualized by staining the gel with Coomassie blue. For quantitative analysis, gels were scanned for densitometric analysis by using the ChemiDoc XRS system (Bio-Rad, Segrate, Italy) to calculate the percentage of ␣⬘-chain. The data obtained were normalized by considering 100% ␣⬘-chain to be equal to the ␣⬘-chain intensity obtained in the absence of factor I (control). The binding data were fit using nonlinear regression analysis (GraFit; Erithacus Software, London, United Kingdom) and a four-parameter logistic analysis was performed to identify the best-fit 50% inhibitory concentration value (the concentration of VCP causing 50% ␣⬘-chain cleavage). Measurement of C3 convertase decay-accelerating activity. The classical pathway decay-accelerating activity of VCP and its mutants was determined by forming EAC142 (37). In brief, the classical pathway C3 convertase EAC142 was made by incubating 100 ␮l of EA (4 ⫻ 109/ml) in DGVB⫹⫹ with 4 ␮g C1 for 20 min at 30°C in a total volume of 120 ␮l. The cells were then washed with ice-cold DGVB⫹⫹, resuspended in 100 ␮l DGVB⫹⫹, and incubated for 20 min at 30°C with 8 ␮g of C4. After the incubation, they were mixed with 4 ␮g of C2 and further incubated for 4 min at 30°C to form EAC142. The convertase formation was then stopped by adding EDTA to a final concentration of 10 mM. To define the effect of VCP and its mutants on the decay of EAC142, 10 ␮l of EAC142 (109/ml) was mixed with various concentrations of VCP or its mutants in a total volume of 25 ␮l for 5 min at 22°C. The reaction mixture was then mixed with 125 ␮l of DGVB and 100 ␮l of guinea pig serum diluted 1:100 in DGVB containing 40 mM EDTA, incubated 30 min at 37°C, and centrifuged. The percentage of lysis was determined by measuring the absorbance at 405 nM.

J. VIROL. The alternative pathway C3 convertase decay was measured by forming C3b,Bb on rabbit (ER) or sheep (ES) erythrocytes. ERC3b and ESC3b were generated by incubating ER or ES with C3, factor B, and factor D in the presence of NiCl2 as previously described (43, 47). C3b,Bb on ER or ES was made by mixing 150 ␮l of C3b-coated rabbit or sheep erythrocytes (109/ml) with 45 ␮g of factor B and 0.76 ␮g of factor D in 210 ␮l of GVB containing 3.5 mM NiCl2. The reaction mixture was then incubated for 5 min at 30°C and stopped by the addition of EDTA to a final concentration of 10 mM. Immediately thereafter, 10 ␮l of ERC3b,Bb or ESC3b,Bb (109/ml) was mixed with various concentrations of VCP or its mutants in a total volume of 50 ␮l and incubated for 10 min at 37°C. The reaction mixture was then mixed with 50 ␮l of human serum diluted 1:5 in GVB containing 20 mM EDTA and incubated at 37°C for 20 min. After incubation, the reaction mixture was mixed with 200 ␮l of GVBE and centrifuged. The percentage of lysis was determined as above. The data generated were normalized by setting 100% lysis equal to the lysis that occurred in the absence of an inhibitor.

RESULTS Design, expression, purification, and characterization of VCP deletion mutants. Three aspects were taken into consideration while designing the mutants. First, since VCP is a soluble protein, mutants were expressed as soluble proteins and not as membrane-bound fusion proteins as described in an earlier study, where attempts were made to localize the C3bbinding site (45). Second, it has previously been shown for the human complement regulators that apart from the CCP domains, the interdomain linker regions may also play a role in the ligand binding (14); therefore, each construct started with the first Cys of the starting domain and ended with the last residue of the interdomain linker. This design kept the entire linker region at the C-terminal side of each mutant (see the amino acid sequences listed in Table 1). Third, several previous studies on the human regulators have shown that two or three contiguous domains (5, 14, 19), as well as their native configuration (i.e., distance between the successive domains) (4, 24), is essential for their complement regulatory activities; therefore, mutants designed contained either two or three successive CCP domains. Earlier, we and others have shown that the Pichia expression system produces a high yield of properly folded VCP (36, 46). Thus, we utilized the same expression system for expressing VCP mutants. The culture supernatants containing the expressed mutants were purified by using a series of chromatographic procedures as described in Materials and Methods. The purified mutants were homogeneous and ⬎95% pure as determined by SDS-PAGE analysis (Fig. 1). To confirm the identity of the mutants, they were subjected to sequencing by mass spectrometry. The amino acid sequences of the expressed mutants were consistent with the predicted sequences confirming the identity of each mutant (Table 1). The addition of EAEAEF in the beginning of each mutant was the result of cloning into the Pichia vector. The molecular masses of the expressed mutants were analyzed by SDS-PAGE and mass spectrometry. In SDS-PAGE, all the proteins migrated higher than their molecular weights; however, this was not due to differences in glycosylation because the primary sequence of VCP does not contain any glycosylation site (23). The data obtained using mass spectrometry confirmed that the molecular masses of the mutants were similar to the calculated masses (Table 1). Characterization of factor I cofactor activity of VCP mutants. A fluid-phase assay was utilized to characterize the fac-

12385 FUNCTIONAL DOMAINS OF VCP VOL. 79, 2005

CCP1-3e

VCP mutant

Amino acid sequencea

13,238.5

13,367.5

14,769.6

19,793.7

20,887.3

Formulab

20,500

26,500

20,500

27,500

26,500

SDS-PAGEc

13,234

13,359

14,712

19,793

20,990

Mass spectrometryd

Mol wt by:

tor I cofactor activity of VCP mutants for C3b and C4b. In this assay, C3b or C4b was incubated with the mutant proteins and factor I, and cleavage of the ␣⬘-chain was assessed by running the samples on SDS-PAGE gels. VCP and the CCP 1-3 mutant supported cleavage of the ␣⬘-chain of C3b (Fig. 2), as well as C4b (Fig. 3), but none of the other mutants showed any detectable activity. These data indicate that CCPs 1 to 3 comprise the minimum region required for the factor I cofactor activity for both C3b and C4b. Incubation of the CCP 1-3 mutant with C3b and factor I did not result in complete cleavage of the ␣⬘-chain of C3b (Fig. 2). We therefore sought to analyze the relative factor I cofactor activity of VCP and the CCP 1-3 mutant for C3b and C4b. In this assay, C3b or C4b was incubated with factor I and increasing concentrations of either

a Boldface amino acid residues indicate residues that are added at the N terminus due to cloning. Underlined sequences indicate sequences determined by mass spectrometry (see Materials and Methods). b Based on the primary sequence. c Calculated from the results shown in Fig. 1. d Determined as described in Materials and Methods. e VCP mutants CCP 1-3 and CCP 1-2 showed two N termini (EAEAEF and EAEF), which was also reflected in their molecular weights determined by mass spectrometry. In the Pichia expression system, the processing of signal sequence occurs in two steps: kex2 cleavage occurs between Arg-Glu, and then STE3 cleaves Glu-Ala repeats. Therefore, the two N termini in these mutants were due to partial N-terminal processing by STE3.

CCP 3-4

CCP 2-3

CCP1-2e

CCP 2-4

EAEAEFCCTIPSRPINMKFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRR CPSPRDIDNGQLDIGGVDFGSSITYSCNSGYHLIGESKSYCELGSTGSMVWNPEAPICESVKCQSPPSISNG RHNGYEDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSDPPTCQIVK EAEAEFCPSPRDIDNGQLDIGGVDFGSSITYSCNSGYHLIGESKSYCELGSTGSMVWNPEAPICESVKCQSPPS ISNGRHNGYEDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSDPPTCQIVKCPHPTISNGYLSSGFKRSY SYNDNVDFKCKYGYKLSGSSSSTCSPGNTWKPELPKCVR EAEAEFCCTIPSRPINMKFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTGTGWTLFNQCIKRR CPSPRDIDNGQLDIGGVDFGSSITYSCNSGYHLIGESKSYCELGSTGSMVWNPEAPICESVK EAEAEFCPSPRDIDNGQLDIGGVDFGSSITYSCNSGYHLIGESKSYCELGSTGSMVWNPEAPICESVKCQSPPS ISNGRHNGYEDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSDPPTCQIVK EAEAEFCQSPPSISNGRHNGYEDFYTDGSVVTYSCNSGYSLIGNSGVLCSGGEWSDPPTCQIVKCPHPTISNG YLSSGFKRSYSYNDNVDFKCKYGYKLSGSSSSTCSPGNTWKPELPKCVR

TABLE 1. Molecular mass and sequence analyses of VCP mutants

FIG. 1. Schematic diagram of VCP and SDS-PAGE analysis of purified recombinant VCP and its various deletion mutants. (Top) Schematic representation of the structure of VCP. It is entirely composed of four complement CCPs. The CCP domains are numbered, and the domain(s) important for binding to C3b and C4b, factor I cofactor activities (CFA) for C3b and C4b, decay-accelerating activity (DAA) for the classical pathway (CP) and alternative pathway (AP) C3 convertases, and binding to heparin are identified. Identification of domains important for various activities except heparin binding (9) is based on the data presented in the manuscript. (Bottom) Purified recombinant full-length VCP and the deletion mutants were subjected to electrophoresis through a 12% SDS-PAGE gel under reducing conditions and stained with Coomassie blue.

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FIG. 2. Analysis of factor I cofactor activity of VCP deletion mutants for C3b. Cofactor activity of the deletion mutants was compared by incubating C3b with VCP (2 ␮g) or its deletion mutants (2 ␮g) in the absence or presence of factor I. The reactions were performed for 4 h and 8 h at 37°C in 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl. The reactions were stopped by the addition of SDSPAGE sample buffer containing DTT and run on 9% SDS-PAGE gels. The cleavages were visualized by staining gels with Coomassie blue. During C3b cleavage, the ␣⬘-chain is cleaved into N-terminal 68-kDa and C-terminal 43-kDa fragments; appearance of these fragments indicates the generation of inactive C3b.

FIG. 3. Analysis of factor I cofactor activity of VCP deletion mutants for C4b. Cofactor activity of the deletion mutants was compared by incubation of C4b with VCP (2 ␮g) or the mutants (2 ␮g) in the absence or presence of factor I. The reactions were performed for 4 h and 8 h at 37°C with 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl and were stopped by the addition of SDS-PAGE sample buffer under reducing conditions. The samples were run on 10% SDSPAGE gels, and the cleavages were visualized by being stained with Coomassie blue. During C4b cleavage, the ␣⬘-chain was cleaved into N-terminal 27-kDa, C-terminal 16-kDa (not visualized on the gel), and central C4d fragments; these cleavages resulted in inactivation of C4b and generation of C4c (composed of 27-kDa and 16 kDa fragments and ␤- and ␥-chains linked together by disulfide bonds) and C4d.

VCP or CCP 1-3 mutant. Figure 4 shows that higher concentrations of the CCP 1-3 mutant than that of VCP were required to cleave the ␣⬘-chains of C3b and C4b. The concentrations of VCP required to cleave 50% of the ␣⬘-chain of C3b and C4b were 0.12 ␮M and 0.038 ␮M, respectively, while the concentrations of the CCP 1-3 mutant required to cleave 50% of the ␣⬘-chain of C3b and C4b were 4.9 ␮M and 0.58 ␮M, respectively. These data indicate that in comparison to VCP, the CCP 1-3 mutant has 41- and 15-fold-lower factor I cofactor activity for C3b and C4b, respectively. Characterization of decay-accelerating activity of VCP mutants. A previous study had demonstrated that apart from factor I cofactor activity, VCP also possesses significant decayaccelerating activity for the classical and limited decay-accelerating activity for the alternative pathway C3 convertases (29). To identify the domains required for the decay-accelerating activity, we measured the activity of VCP mutants using he-

molytic assays. For measuring the decay-accelerating activity for the classical pathway C3 convertase C4b,2a, the enzyme was formed on the sensitized sheep erythrocytes and allowed to incubate with the mutant proteins. The remaining C3 convertase activity was quantitated by measuring hemolysis after adding EDTA sera (a source of C3 to C9). VCP showed significant decay-accelerating activity even at a 100 nM concentration. On a molar basis, VCP was only 1.8-fold-less active than sCR1. Among the mutants, CCP 1-3 and CCP 1-2 mutants showed decay-accelerating activity, while the other mutants did not show any activity (Fig. 5), indicating that CCPs 1 to 2 comprise the minimum region required for the classical pathway. It should be pointed out here that the concentration of CCP 1-2 mutant required to inhibit the decay-accelerating activity was 5,600-fold higher than the concentration of VCP (Fig. 5), suggesting that in addition to CCPs 1 to 2, CCPs 3 and

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FIG. 4. Comparison of factor I cofactor activity of VCP and the CCP 1-3 mutant for C3b and C4b. C3b (left) or C4b (right) and factor I were incubated in 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl with increasing concentrations of VCP or the CCP 1-3 mutant at 37°C for 4 h. Cleavage products were visualized by running the samples on 9% and 10% SDS-PAGE gels for C3b and C4b, respectively, and staining with Coomassie blue. The intensity of ␣⬘-chains of C3b and C4b was determined by densitometric analysis and represented graphically (bottom).

4 also contribute significantly to the classical pathway decayaccelerating activity. For measuring the alternative pathway decay-accelerating activity of VCP and its mutants, the alternative pathway C3 convertase C3b,Bb was formed on rabbit erythrocytes. The enzyme was incubated with VCP or the mutant proteins, and the remaining enzyme activity was detected by adding EDTA sera and measuring hemolysis. Surprisingly, neither VCP nor its mutants showed any alternative pathway decay-accelerating activity even at a 50 ␮M concentration. In a parallel assay, factor H and sCR1 showed significant inhibition at 100 nM and 2.5 nM concentrations, respectively (Fig. 6A). Since the previ-

ous study which reported limited alternative pathway decayaccelerating activity of VCP utilized sheep erythrocytes for forming C3b,Bb (29) and not the rabbit erythrocytes, we tested if the lack of inhibition in our assays was due to the differences in the cells used. Thus, we formed C3b,Bb on sheep erythrocytes and measured the decay-accelerating activity of VCP and its mutants. On sheep cells, VCP accelerated the decay of alternative pathway C3 convertase, though the concentration of VCP required to decay 50% alternative pathway C3 convertase was 64 ␮M, which was 2,400-fold higher than that required to decay the classical pathway C3 convertase (Fig. 6B). Among VCP mutants, only the CCP 2-4 mutant showed

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FIG. 5. Classical pathway decay-accelerating activity of VCP mutants. The classical pathway (CP) C3 convertase C4b,2a was formed on antibody-coated sheep erythrocytes by using purified C1, C4, and C2 (Calbiochem). The cells coated with C4b,2a were incubated with increasing concentrations of VCP or its mutants at 22°C for 5 min, and the remaining C3 convertase activity was assayed by incubation of the cells with guinea pig sera diluted 1:100 in dextrose gelatin veronal buffer, pH 7.4, containing 40 mM EDTA. sCR1 and factor H were used as controls. The data were normalized by setting 100% C3 convertase activity to be equal to the average activity in the absence of any inhibitor (VCP or its mutants).

inhibition, although like VCP at a similarly high concentration, suggesting that the limited AP decay-accelerating activity is imparted by CCPs 2 to 4. Effect of membrane sialic acid on the decay-accelerating activity of VCP. From the data presented in Fig. 6, it is clear that VCP shows limited decay-accelerating activity against the alternative pathway C3 convertase present on sheep erythrocytes but not against the convertase present on rabbit erythrocytes. Previously, differences in the decay-accelerating activity against the alternative pathway C3 convertase present on sheep versus rabbit erythrocytes have been reported for human complement regulator factor H. It has been shown that factor H displays enhanced decay-accelerating activity towards the alternative pathway C3 convertase present on sheep erythrocytes; this has been attributed to the interaction of its heparinpolyanion-binding site with sialic acid present on the erythrocyte membrane (rabbit erythrocytes contain approximately 13% of the sialic acid present on sheep erythrocytes) (8, 40). Since VCP contains a heparin-binding site on CCP 4 (9), we sought to analyze if the differences in its decay activity on sheep versus rabbit erythrocytes were due to interaction of its heparin-polyanion-binding site with sialic acid present on sheep erythrocytes. For this purpose, we treated sheep erythrocytes with neuraminidase and compared the decay-accelerating activity of VCP against the C3 convertase present on untreated and treated erythrocytes; factor H was included as a positive control. As expected, factor H showed reduced alternative pathway C3 convertase decay-accelerating activity on neuraminidase-treated cells compared to untreated cells (Fig. 7); the decay activity was 7.4-fold less on the treated than on the untreated sheep cells. However, VCP did not show any difference in the decay activity, thereby indicating that like factor H, the heparin-binding site on VCP does not help in

enhancing the decay of the alternative pathway C3 convertase on sheep cells. Thus, the observed differences in the decay activities of VCP on sheep and rabbit erythrocytes were not due to the differences in the membrane sialic acid content. Binding of VCP mutants to C3b and C4b. From the data presented above, it is clear that CCPs 1 to 2 and 2 to 4 are the minimum regions required for the decay-accelerating activities and that CCPs 1 to 3 are required for cofactor activities. To determine whether reduced decay-accelerating activities and cofactor activities of the VCP mutants correlate with binding to C3b and C4b, we performed a quantitative analysis of binding of VCP and its mutants to C3b and C4b using a surface plasmon resonance assay. In this assay, C3b and C4b were oriented on a streptavidin chip by labeling their free -SH groups with biotin (2). This setup mimicked the physiological orientation of these proteins and also allowed accurate determination of the binding constants (2). When VCP or its deletion mutants were allowed to flow over the sensor chip, only VCP and the CCP 1-3 mutant bound to C3b and C4b (Fig. 8). Further analysis showed that the CCP 1-3 mutant had 36- and 228-fold-lower affinity for C3b and C4b, respectively, than VCP (Fig. 9 and Table 2). This decrease was primarily due to reduction in the on-rates of the mutant (Table 2). These data suggest that poor factor I cofactor activities of the CCP 1-3 mutant could be due to its lower affinity for C3b and C4b. It is, however, interesting that the CCP 1-2 and CCP 2-4 mutants, which showed the classical and alternative pathway decay-accelerating activities, respectively, showed no detectable binding to C4b and C3b. DISCUSSION In the present study, we utilized a series of VCP deletion mutants to address the contribution of each CCP domain to its

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FIG. 6. Alternative pathway decay-accelerating activity of VCP and its mutants. The alternative pathway (AP) C3 convertase C3b,Bb was formed on rabbit (ER) (A) or sheep (ES) (B) erythrocytes using purified C3 and factors B and D in the presence of NiCl2 (29, 43). The cells coated with C3b,Bb were incubated with increasing concentrations of VCP or its mutants at 37°C for 10 min, and the remaining C3 convertase activity was assayed by incubation of the cells with human sera diluted 1:5 in gelatin veronal buffer, pH 7.4, containing 20 mM EDTA. Factor H and sCR1 were used as positive controls. The data were normalized by setting 100% C3 convertase activity to be equal to the average activity in the absence of inhibitor (VCP or its mutants).

cofactor activities, decay-accelerating activities, and binding to C3b and C4b. Our data on the localization of domains required for factor I cofactor activity for C3b and C4b indicate that a minimum of three CCPs (CCPs 1 to 3) are essential for these activities, because further deletion of either CCP 1 or CCP 3

completely abolished the cofactor activities (Fig. 2 and 3). These data are consistent with the previous observations on mapping of the human RCA proteins (factor H, C4bp, MCP, and CR1) for cofactor activities, which indicated that a minimum of three CCPs are required for cofactor activities (4, 11,

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FIG. 7. Comparison of alternative pathway decay-accelerating activity of VCP and its mutant CCP 2-4 on untreated and neuraminidase-treated sheep erythrocytes. Sheep erythrocytes (2 ⫻ 109) in 50 mM sodium acetate, pH 5.5, containing 154 mM NaCl and 68 mM CaCl2 were treated with 0.1 U of neuraminidase (Roche Diagnostics, Mannheim, Germany) at 37°C for 90 min. This treatment removed 72 to 74% of sialic acid as measured by the thiobarbituric acid assay for sialic acid (58). The alternative pathway (AP) C3 convertase C3b,Bb was formed on the untreated or neuraminidase-treated sheep erythrocytes (Es) using purified C3 and factors B and D in the presence of NiCl2, and the decay activity was measured as described in Materials and Methods. Factor H was used as a control. The data were normalized by setting 100% C3 convertase activity to be equal to the average activity in the absence of inhibitor.

14, 16, 19, 20, 44, 52). There is now a consensus that factor I-mediated inactivation of C3b and C4b involves two steps: (i) recognition of C3b or C4b by the cofactor and (ii) interaction of factor I with C3b or C4b and the cofactor (48, 54). Thus, it is conceivable that the recognition sites for C3b or C4b and factor I on the complement regulators are spatially conserved and require three CCPs. This, however, does not seem to be a general phenomenon as our recent data on kaposica, the complement regulator of the Kaposi’s sarcoma-associated herpes-

virus, indicated that only two CCPs are enough to impart cofactor activities in kaposica (35). Since CCPs 1 to 3 are required for both C3b and C4b cofactor activities, it can be envisaged that the recognition site for C3b and C4b on VCP is formed by similar residues. This possibility, however, appears to be less likely for VCP as it binds to different regions on C3b and C4b; its binding site on C3b is located on the C3dg region, while its binding site on C4b is located on the C4c region (2). VCP has previously been shown to possess efficient decay-

FIG. 8. Binding of VCP mutants to C3b and C4b. Binding was determined by SPR. Sensograms were generated by injection of VCP (0.6 ␮M) or its mutants (40 ␮M) over a streptavidin sensor chip immobilized with C3b (Fc-2) and C4b (Fc-3). Fc-1 served as a control flow cell. (A) Sensograms depicting binding to C3b; (B) sensograms depicting binding to C4b.

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FIG. 9. Analysis of binding of VCP and the CCP 1-3 mutant to immobilized C3b and C4b. Various concentrations of VCP or the CCP 1-3 mutant were injected over a streptavidin chip, wherein Fc-1served as a control flow cell and Fc-2 and Fc-3 were immobilized with C3b and C4b, respectively. (Left) Sensogram overlays for binding of VCP and the CCP 1-3 mutant to immobilized C3b; (right) sensogram overlays for binding of VCP and the CCP 1-3 mutant to immobilized C4b. The wavy lines represent the sensograms obtained; the straight lines represent the global fitting of the data to a 1:1 Langmuir binding model (A ⫹ B 7 AB; BIAevaluation 4.1). The concentration of analytes injected is indicated at the right of each sensogram overlay.

accelerating activity for the classical pathway and weak decay activity for the alternative pathway C3 convertases (29). Our data show that CCPs 1 to 2 of VCP form the minimum region essential for its classical pathway decay-accelerating activity, but CCPs 3 and 4 are required for its complete activity (Fig. 5). Earlier studies of DAF showed that CCPs 2 to 3 are important for its classical pathway decay-accelerating activity (5). It is pertinent to point out here that CCPs 1 to 2 of VCP show maximum sequence similarity to CCPs 2 to 3 of DAF, which possesses classical pathway decay-accelerating activity (5). Our data show that VCP possesses limited decay-accelerating activity only against the alternative pathway C3 convertase present on the sheep erythrocytes and not against the C3 convertase present on rabbit erythrocytes (Fig. 6). Since VCP contains a heparin-binding site like factor H (9), it is likely that

similar to factor H (31, 49), the heparin-binding site on VCP helps it to enhance its decay-accelerating activity against the alternative pathway C3 convertase present on the sheep erythrocytes, which contains a high surface density of sialic acid. However, this premise did not hold true, as the decay rate of VCP did not reduce on neuraminidase-treated sheep erythrocytes (Fig. 7). The weak decay activity was also observed with the CCP 2-4 mutant, suggesting that CCPs 2 to 4 are important for this activity. Like VCP, the Kaposi’s sarcoma-associated herpesvirus complement control protein kaposica also contains very weak decay-accelerating activity against alternative pathway C3 convertase (35, 55). Thus, it seems that viral homologs of complement control proteins primarily contain three out of four C3 convertase regulatory activities of the host complement regulators.

TABLE 2. Kinetic and affinity data for the interactions of VCP and the CCP 1–3 mutant with C3b and C4ba Ligand

C3b C3b C4b C4b C3b C3b C4b C4b a b c

Analyte

kd(1/s)/ka(1/Ms)

VCP VCP VCP VCP CCP 1-3 CCP 1-3 CCP 1-3 CCP 1-3

1.57/9.07 ⫻ 10 NA 0.309/1.61 ⫻ 106 NA 0.13/2.08 ⫻ 103 NA 0.363/8.27 ⫻ 103 NA

KDa (␮M)

SE (kd/ka) 5

⫺1

1.56 ⫻ 10 /9.58 ⫻ 10 NA 6.56 ⫻ 10⫺3/3.52 ⫻ 104 NA 9.59 ⫻ 10⫺3/269 NA 0.0102/266 NA 4

b

1.73 1.94c 0.193b 0.185c 62.4b 68.4c 43.9b 42.3c

NA, not applicable; ka, association rate constant; kd, dissociation rate constant; KD, equilibrium rate constant; SE, standard error. Data were calculated by global fitting to a 1:1 Langmuir binding model (BIAevaluation 4.1). Data were calculated by steady-state analysis (BIAevaluation 4.1).

␹2

0.327 0.521 0.775 1.52 0.131 0.0633 0.236 0.109

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Our data on binding of VCP to target proteins measured using SPR identified CCPs 1 to 3 as the smallest structural unit capable of binding to C3b and C4b (Fig. 8). The presence of CCP 4, however, significantly enhanced the affinities for C3b and C4b (Fig. 9 and Table 2). These findings are consistent with the previous data on binding of the human RCA proteins to C3b and C4b, which essentially suggested that C3b or C4b binding primarily resides within three or four CCPs (4, 14, 19, 20, 52). Previously, two studies attempted to localize the C3bbinding site on VCP. Both these studies suggested that all the four CCPs are required for C3b binding. In the first study, various domains of VCP were expressed as membrane-bound CR2 fusion proteins (45). The lack of binding of the CCP 1-3 mutant to C3b in this study (as opposed to our present study) could have been due to lower sensitivity of the assay used. Alternatively, it is possible that the fusion of CCP 3 to CR2 could have sterically hindered the access to residues present on CCP 3, which are critical for binding to C3b. Unlike the first study, the second study utilized soluble proteins and SPR technology for detecting binding but failed to test binding of the CCP 1-3 mutant to C3b due to lack of this mutant (53). Even though ligand binding is a prerequisite for cofactor and decay activities, a large body of evidence indicates that ligand binding does not always correlate well with cofactor and decay activities (26, 41). Our data support this premise. For example, the CCP 1-3 mutant showed a 36-fold decrease in affinity for C3b and a 228-fold decrease in affinity for C4b compared to VCP (Table 2). Although the decrease in C3b binding of the CCP 1-3 mutant correlated well with a 41-fold reduction in its C3b cofactor activity, the dramatic reduction in its affinity for C4b did not correlate with the 15-fold decrease in C4b cofactor activity. Similarly, CCP 1-2 and CCP 2-4 mutants which exhibited the classical and alternative pathway decay-accelerating activities, respectively, did not show detectable binding to C4b and C3b. It is likely that like DAF, these mutants might have better affinity for the C3 convertases than for their subunits C4b and C3b; DAF has been shown to bind to C4b,2a with 1,000-fold higher affinity than to C4b alone (38). The data discussed above point out that although only two or three CCPs are enough to mediate the cofactor and decay activities of VCP, the entire length of the protein is essential for its full functional activity. It is likely that the residues scattered on two or three CCPs together form a binding site for C3 convertases or their subunits (C3b or C4b) and that the other CCPs contribute to the conformational stability of the complexes. Alternatively, a larger surface spanning all the four domains of VCP contributes to forming the recognition site for the convertases and C3b or C4b. These possibilities assume that functional domains are arranged in a linear fashion. This, however, may not be true; it is also likely that the recognition sites are discontinuous and that the middle domains or regions serve as essential spacers. These data, along with our recent observation on kaposica, which also indicated that the entire length of the protein is important for its complete function (35), explain why the four-CCP structure of viral CCP is highly conserved in viruses. The important question in vaccinia virus biology is how VCP helps to protect the vaccinia virus from complement-mediated neutralization in vivo. The complement system is known to neutralize viruses in the presence and the absence of specific

J. VIROL.

antibodies. Previously, it has been shown that extracellular enveloped virus, which is important for the virus dissemination, is protected from the alternative pathway due to the presence of host complement regulators on its envelope (57). However, this form remains susceptible to classical pathway-mediated neutralization (57). Thus, vaccinia virus must encode an effective regulator against the classical pathway to overcome the classical pathway-mediated neutralization. Earlier studies have shown VCP to be a better regulator of the classical pathway than even the host complement regulators factor H and C4binding protein (21, 46); in the present manuscript, we have shown that VCP is only twofold-less effective than sCR1 in decaying the classical pathway C3 convertase (Fig. 5). The question is whether this inhibition of the classical pathway is also seen in vivo? In an earlier in vivo study, it was shown that the lesions formed by the VCP deletion mutant and the wildtype virus were similar for the first few days, but the lesion size was significantly reduced after 5 days in the mutant virus (15). Further, the appearance of antibody capable of mediating complement-enhanced neutralization correlated well with this timing (15), suggesting that VCP effectively inhibits classical pathway-mediated neutralization in vivo. In the present manuscript, we have shown which of the domains of VCP are required for each of its complement regulatory activities. Further delineation of the vital control points in VCP important for inhibition of the classical pathway decay-accelerating activity and C4b cofactor activity and targeting these control points would allow evaluation of the relative importance of these activities in immune evasion and may provide an alternative approach to manage vaccinia virus vaccine-related complications. ACKNOWLEDGMENTS We thank John D. Lambris (Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA) for his continuous support; Michael K. Pangburn (Department of Biochemistry, University of Texas Health Science Center, Tyler, Texas) for his support and the generous gift of complement proteins factors B, D, H, and I; Henry Marsh (AVANT Immunotherapeutics, Inc., Needham, Mass.) for providing sCR1; Nicholas E. Sherman (Biomolecular Research Facility, University of Virginia, Charlottsville, Va.) for sequencing VCP mutants; and Gabriela Canziani (Protein Interaction Facility, University of Utah, Salt Lake City, Utah) for advice on maintenance of Biacore. This work was supported by a Wellcome Trust Senior Research Fellowship in Biomedical Science in India to A.S. The authors also acknowledge financial assistance to A.K.S. by the Council of Scientific and Industrial Research of India. REFERENCES 1. Alcami, A., and U. H. Koszinowski. 2000. Viral mechanisms of immune evasion. Trends Microbiol. 8:410–418. 2. Bernet, J., J. Mullick, Y. Panse, P. B. Parab, and A. Sahu. 2004. Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b. J. Virol. 78:9446–9457. 3. Bernet, J., J. Mullick, A. K. Singh, and A. Sahu. 2003. Viral mimicry of the complement system. J. Biosci. 28:249–264. 4. Blom, A. M., L. Kask, and B. Dahlback. 2001. Structural requirements for the complement regulatory activities of C4BP. J. Biol. Chem. 276:27136– 27144. 5. Brodbeck, W. G., D. Liu, J. Sperry, C. Mold, and M. E. Medof. 1996. Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor. J. Immunol. 156:2528–2533. 6. Carroll, M. C. 2004. The complement system in regulation of adaptive immunity. Nat. Immunol. 5:981–986. 7. Cooper, N. R. 1998. Complement and viruses, p. 393–407. In J. E. Volanakis and M. M. Frank (ed.), The human complement system in health and disease. Marcel Dekker, Inc., New York, N.Y.

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