Functional Activity of the Complement Regulator Encoded by Kaposi's Sarcoma-associated Herpesvirus*

O. Brad SpillerDagger §, David J. Blackbourn, Linda Mark||, David G. ProctorDagger , and Anna M. Blom||**

From the Dagger  University of Wales College of Medicine, Virus Receptor and Immune Evasion Group, Department of Medical Biochemistry, Heath Park, Cardiff CF14 4XX, United Kingdom, the  Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom, and the || Department of Clinical Chemistry, Lund University, University Hospital Malmö, Malmö S-205 02 Sweden

Received for publication, November 13, 2002, and in revised form, January 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma and certain B-cell lymphomas. The fourth open reading frame of the KSHV genome encodes a protein (KSHV complement control protein (KCP, previously termed ORF4)) predicted to have complement-regulating activity. Here, we show that soluble KCP strongly enhanced the decay of classical C3-convertase but not the alternative pathway C3-convertase, when compared with the host complement regulators: factor H, C4b-binding protein, and decay-accelerating factor. The equilibrium affinity constant (KD) of KCP for C3b and C4b was determined by surface plasmon resonance analysis to range between 0.47-10 µM and 0.025-6.1 µM, respectively, depending on NaCl concentration and cation presence. Soluble and cell-associated KCP acted as a cofactor for factor I (FI)-mediated cleavage of both C4b and C3b and induced the cleavage products C4d and iC3b, respectively. In the presence of KCP, FI further cleaved iC3b to C3d, which has never been described before as complement receptor 1 only mediates the production of C3dg by FI. KCP would enhance virus pathogenesis through evading complement attack, opsonization, and anaphylaxis but may also aid in targeting KSHV to one of its host reservoirs since C3d is a ligand for complement receptor 2 on B-cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kaposi's sarcoma-associated herpesvirus (KSHV)1 is the likely etiologic agent of Kaposi's sarcoma and is the most recently identified member of the human Herpesviridae family (1, 2). KSHV is also associated with the B-cell tumors body cavity-based primary effusion lymphomas and the plasma cell variant of multicentric Castleman's Disease (for reviews, see Refs. 3 and 4). KSHV belongs to the rhadinovirus genus of the Gammaherpesvirinae subfamily, the prototype of which is Herpesvirus saimiri (HVS). The long unique region of the KSHV genome comprises 140.5 kb and contains over 80 open reading frames (ORFs) (5). Several of the ORFs encode host cell homologues (e.g. viral cyclin D, viral interleukin-8 G protein-coupled receptor, and a bcl-2 homologue) with the potential to regulate the cell cycle and the immune response thereby contributing to the virulence and the pathogenesis of KSHV (5, 6).

The fourth open reading frame, ORF4, was initially speculated to have complement regulatory abilities based on its homology to human complement regulators decay-accelerating factor (DAF) and membrane cofactor and to previously described virus-encoded complement inhibitors (5). The KSHV ORF4 gene is predicted to encode a KCP protein of 550 amino acids (data base reference SPTREMBL:O40912), and the first 280 amino acids are predicted to encode four complement control protein (CCP) domains. CCP domains are defined by a consensus sequence of ~60 amino acids containing four invariant cysteine residues that form disulfide links, which results in the CCP forming a globular domain with a hydrophobic core enclosed by beta -strands (7, 8). While the CCP domain is not exclusive to complement control proteins, all but one of the C3-convertase-regulating proteins identified to date, either mammalian or viral, are composed of CCPs. Analyses of KCP mRNA expressed in B-cells isolated from primary effusion lymphoma patients revealed three major transcript variants (one unspliced full-length form and two alternatively spliced variants), all containing the N-terminal four CCP domains but with different amounts of internal coding sequence removed by alternative splicing (9). All three transcripts also encode the transmembrane region indicating that all forms are membrane-associated. The HVS-encoded complement regulator is also alternatively spliced, but the transmembrane region is lost in the single alternatively spliced form due to a frameshift (10). The complement regulator homologue encoded by murine gammaherpesvirus 68 (Mgamma HV68) has also been reported to be expressed as cell-bound and soluble forms (11); however, no alternate splicing of the Mgamma HV68 mRNA that could yield the latter form was observed by the authors, and how the soluble protein arises has yet to be resolved.

During our initial characterization of the KCP mRNA isolated from primary effusion lymphoma cell lines, we found that expression of the KCP cDNA in CHO cells significantly reduced C3b deposition on antibody-sensitized CHO cells following incubation with whole human serum; however, the mechanism of complement regulation was not elucidated (9). Here we describe the mechanism of KCP complement regulation. We examined the ability of KCP to enhance the decay of C3-convertases of both the alternative and classical pathways of complement. These activities were compared with those of the host complement regulators, factor H (FH), C4-binding protein (C4BP), and DAF. The ability of KCP to act as a factor I (FI) cofactor for the cleavage and inactivation of both C3b and C4b was also examined in the soluble phase and on the surface of cells. Importantly, we show that KCP has FI cofactor activity previously undescribed as it promotes FI production of the C3d fragment.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins-- Human C4BP (12), C1 (13), C2 (14), C4 (15), FH (16), and FI (17) were purified from plasma as described previously. C3, C3b, C4b, factors B, D, and properdin were purchased from Advanced Research Technologies. C1 and C2 were functionally pure (i.e. they were devoid of other complement factors), while C4BP, FH, FI, C4, C3, C4b, and C3b were at least 95% pure as determined by Coomassie staining of proteins separated by SDS-PAGE. All proteins were stored in aliquots at -80 °C. Protein concentrations were determined by measuring absorbance at 280 nm. C3b and C4b were labeled with 125I using the chloramine T method (18), and the specific activity was determined to be 0.4-0.5 MBq/µg of protein. Human erythrocyte ghosts were used as a source of CR1 and were made through repeated washing (until all red color was removed) in ice-cold phosphate buffer (7.5 mM phosphate, 1 mM EGTA, 0.2 M phenylmethylsulfonyl fluoride) by resuspension and centrifugation at maximum speed in a benchtop microfuge (4 °C) followed by discarding the supernatant.

Expression and Purification of KCP-- Soluble recombinant Fc fusion proteins were constructed based on the naturally occurring isoforms of KCP (described in Ref. 9) using the same strategy and vectors as previously described to construct DAF-Fc (19). Cloning and characterization of full-length KCP cDNA are described elsewhere (9). Briefly, PCR primers were designed to truncate the recombinant KCP isoforms immediately following the CCP domains (short: 5'-AGCTGCTGTATGGGTGTCTTCA-3'), prior to the Ser/Thr-rich region (medium; 5'-TGGCTGGGATGTAGTTTTCTCAT-3'), or prior to the transmembrane region (long: 5'-AATGGGAGGGAGTGTTGGTTCT-3'). All of these primers were designed with a NotI restriction site to allow the in-frame addition of the human IgG1-Fc C terminus when used for PCR in combination with the common forward primer (F: 5'-GCGCTCTAGAGCTAGCATGGCCTTTTTAAGACAAAC-3'), followed by cloning into the previously described expression vector pDR2Delta EF1alpha (19). The resultant plasmids were transfected into CHO cells (European culture collection) using LipofectAMINE (Invitrogen). Cells were propagated in RPMI 1640 cell medium containing 10% fetal calf serum, and stable transfectants were created by propagating cells in medium containing 400 µg/ml of Hygromycin B (Invitrogen) for 2 weeks. Cell supernatants were collected, and the Fc fusion proteins isolated and purified as previously described using protein A-Sepharose (Amersham Biosciences) (19). The resulting three KCP-Fc fusion proteins of different sizes resemble IgG molecules with the Fab portion replaced with KCP proteins. These proteins were released by incubation with papain-coupled agarose (Sigma) at 37 °C for 1 h (the optimal ratio between amount of the protein and papain was determined by titration). Papain-agarose was then removed by centrifugation, and the free Fc and uncleaved Fc fusion protein removed from the supernatant by binding to a protein A-Sepharose column. The purity of all proteins was confirmed by silver staining following separation by SDS-PAGE, and the protein concentration was determined by measuring absorbance at 280 nm. Cell surface expression of all three KCP fusion proteins was accomplished by exchanging in-frame the human Fc portion of the recombinant proteins with the cDNA encoding the signal for GPI anchoring of human DAF (see Ref. 9). Uniform expression was confirmed by flow cytometry using monoclonal anti-KCP antibodies raised in-house.

C3b/C4b-degradation Assay-- Various concentrations of putative or control cofactor proteins were mixed with 750 nM C3b (or 250 nM C4b), 60 nM FI, and trace amounts of 125I-labeled C3b (or 125I-labeled C4b) in 50 µl of 50 mM Tris-HCl, pH 7.4, supplemented with 150 mM NaCl. For examination of cell-associated KCP cofactor activity the unlabelled C3b/C4b were not included in the incubation. The samples were incubated for 1.5-4 h for soluble proteins or overnight for cells at 37 °C, and the reaction was terminated by the addition of SDS/PAGE sample buffer with reducing agent. The samples were then incubated at 95 °C for 3 min before protein separation on 10-15% gradient polyacrylamide gels and visualization by autoradiography using Phosphorimaging analysis (Molecular Dynamics).

Inhibition of Classical Pathway C3-convertase-- Sheep erythrocytes were washed twice with DGVB2+ (2.5 mM veronal buffer, pH 7.3, 72 mM NaCl, 140 mM glucose, 0.1% gelatin, 1 mM MgCl2, and 0.15 mM CaCl2), suspended at a concentration of 109 cells/ml, and incubated for 20 min at 37 °C with an equal volume of amboceptor (Roche Molecular Biochemicals; diluted 1:3000 in DGVB2+) to make EA cells. EAC1 cells were made by washing the EA cells twice with ice-cold DGVB2+ and resuspending at 109 cells/ml; then C1 was added to 1010 cells drop-wise to a final concentration of 5 µg/ml, and the mixture incubated with agitation for 20 min at 30 °C. EAC1 cells were washed twice with ice-cold buffer and incubated with agitation for 20 min at 30 °C with 1 µg/ml C4. The resultant EAC14 cells were incubated in DGVB2+ containing C2 (~5 µg/ml) for 5 min to allow formation of C3-convertase. The cells were then placed on ice for 1 min, centrifuged, and resuspended in prewarmed DGVB2+. An equal volume of these EAC142 cells was added to a range of prediluted inhibitors and allowed to incubate at 30 °C with constant shaking for 5 min. 100-ml aliquots of each sample were removed and added to 100 µl of guinea-pig serum-diluted 1:50 in 40 mM EDTA-GVB, and the resultant lysis was determined following incubation at 37 °C for 60 min. The amount of released hemoglobin was directly proportional to the residual C3-convertase activity remaining on the EA142 cells and was measured at 405 nm in the supernatant after pelleting the unlysed cells by centrifugation. Soluble recombinant DAF-Fc or purified C4BP were used as positive controls in these experiments, and all inhibitors were compared with the amount of lysis observed in the absence of added inhibitors.

Inhibition of the Alternative Pathway C3-convertase-- Ten ml of EAC14 (1.5 × 109/ml), prepared as described above, were incubated with 0.25 µg/ml C2 and 50 µg/ml C3 for 30 min at 30 °C with agitation. The cells were washed once in 10 mM EDTA-GVB, resuspended in the same buffer, and incubated for 2h at 37 °C to allow dissociation of C1 and C2 from the cells. The resultant EAC43 were washed twice in EDTA-GVB, then twice in Mg2+-EGTA buffer (2.5 mM veronal buffer, pH 7.3, 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 7 mM MgCl2, and 10 mM EGTA), and then resuspended at 2 × 109 cell/ml. EAC43 were then incubated with properdin (0.21 µg/ml), FD (21 ng/ml), FB (10 ng/ml), and tested inhibitor or Mg2+-EGTA (final volume 150 µl). The mixtures were kept shaking at 30 °C and 100-µl aliquots removed after 30 min and added to 100 µl of guinea pig serum diluted 1:30 in GVB-EDTA. After 1 h at 37 °C, the samples were centrifuged, and the amount of erythrocyte lysis was determined spectrophotometrically as above. Soluble recombinant DAF-Fc and FH served as positive controls in these experiments, and all inhibitors were compared with the amount of lysis observed in the absence of added inhibitors.

N-terminal Protein Sequencing-- C3b was treated with FI in the presence of the short form of KCP as described in the degradation assay section. The proteins containing in total 7.5 µg of C3 were separated by SDS/PAGE on 10-15% gradient gel and transferred by semi-dry electroblotting onto polyvinylidene difluoride membrane. The membrane was stained with Coomassie and the appropriate bands were excised and subjected to N-terminal sequencing in an automated sequenator (494 Protein Sequencer ProCise; Applied Biosystems).

Surface Plasmon Resonance (Biacore)-- The interaction between C3b/C4b and KCPs was analyzed using surface plasmon resonance (Biacore 2000; Biacore). Each of four flow cells on a CM5 sensor chip were activated with 20 µl of 0.2 M 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide with 0.05 M N-hydroxy-sulfosuccinimide at a flow rate of 5 µl/min, and then the short form of recombinant KCP (0.01 mg/ml in 10 mM sodium-acetate buffer, pH 4.5) was injected over flow cell 2 to reach 2000 resonance units. Medium and long forms of KCP were injected over flow cells 3 and 4, respectively, to also reach 2000 resonance units. In all cases unreacted groups were blocked with 20 µl of 1 M ethanolamine, pH 8.5. A negative control was prepared by activating and subsequently blocking the surface of flow cell 1.

The association kinetics were studied for a range of C3b and C4b concentrations (methylamine inactivation of C3 and C4 was used to convert native C3 and C4 to C3b and C4b conformation, respectively (20)) using the standard flow buffer (10 mM Hepes-KOH, pH 7.4 supplemented with 150 mM NaCl, 0.005% Tween 20). In some experiments, 75 mM NaCl was used, and occasionally standard flow buffer was supplemented with physiological concentrations of calcium and zinc (2.5 mM CaCl2, 20 µM ZnCl2). Protein solutions were injected for 300 s to achieve saturation during the association phase at a constant flow rate of 30 µl/min. The sample was injected first over the negative control surface and then over immobilized KCP flow cells and analyzed for a dissociation phase of 200 s at the same flow rate. Signals were normalized by subtracting the nonspecific signal measured by control flow cell 1. Between each different concentration of C4b or C3b tested the flow cell surfaces were regenerated with a 30-µl injection of 2 M NaCl to remove bound ligands. All sensograms were analyzed using the BiaEvaluation 3.0 software (Biacore) to calculate equilibrium affinity constants.

Flow Cytometry-- Deposition of C3b and various fragments thereof was measured by flow cytometry using polyclonal antibodies. C3b was deposited in the absence of sensitizing antibody, on the surface of sheep erythrocytes (EC3) after three rounds of incubation with C3, FB, FD, and nickel ions as previously described (16). The cells were then incubated overnight at 37 °C with FI alone or in combination with FH, the short form of KCP, or a source of CR1 (human erythrocyte ghosts) in DGVB2+. Aliquots of each of these treated EC3 cells were then incubated with a panel of rabbit polyclonal antibodies directed against C3d (Dakopatts), C3c (two different antibodies from Dakopatts), or whole C3 molecule (generated in house against whole purified C3). EC3 cells were washed once in flow cytometry medium (FCM; phosphate-buffered saline, 15 mM EDTA, 30 mM NaN3, 1% bovine serum albumin) and resuspended to a concentration of 106 cells/ml. Primary antibodies (purified IgG fractions) were diluted 1/100 in FCM and allowed to bind for 30 min at 4 °C. Unbound antibody was removed by three washes in FCM prior to incubation with secondary fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin antibody (Dakopatts). After three final washes in FCM the cells were analyzed on a flow cytometer (FACSCalibur, BD Biosciences). All treated EC3 cells were compared with EC3 cells that were incubated overnight in DGVB2+ only as a negative control. All analyses were performed in triplicate, and the experiment was performed twice.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of Three Splice Variants of KCP-- Three soluble recombinant Fc fusion forms of KCP were constructed based on the naturally occurring transcript variants observed in KSHV-infected cells (9). The shortest construct is composed of only the N-terminal CCP domains fused with the hinge and C terminus of human IgG1 Fc region. The medium and longest recombinant fusion proteins were truncated prior to the Ser/Thr-rich region or prior to the transmembrane regions, respectively. Fig. 1 shows the molecular mass of all three purified KCP-Fc forms compared with human DAF-Fc separated by SDS-PAGE under reducing conditions. However, it should be noted that Fc fusion proteins appear as IgG molecules with the Fab portion replaced by KCP, and purified KCP-Fc proteins are therefore dimeric. Additionally, because the longest KCP form retained both of the mRNA splice donor sites and the single splice acceptor site, the resultant fusion protein existed as a mixture of full-length, medium, and short Fc fusion proteins.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1.   Analysis of KCP proteins by SDS/PAGE. Purified DAF-Fc and the three recombinant forms of KCP-Fc (~2 µg/well) were separated by SDS/PAGE on 7.5% gel under reducing conditions and then visualized by silver staining. The molecular mass markers are shown to the left.

KCP Accelerates Decay of the Classical Pathway C3-convertase-- The decay of classical pathway C3-convertase was measured in the presence of soluble KCP (Fig. 2). Putative or known inhibitory proteins were added after the formation of the classical C3-convertase on the surface of sheep erythrocytes, and decay acceleration was measured after 5 min. All three forms of KCP-Fc accelerated the decay of C3-convertase equivalently and were ~10-fold less active than the positive control DAF-Fc (Fig. 2A). Monomeric KCP forms were released from KCP-Fc with papain and found to be about 5-fold less active than C4BP at enhancing the decay (Fig. 2B). Dimeric KCP-Fc was found to be equally as effective as monomeric KCP, indicating that the Fc fusion proteins did not cause steric hindrance of the active site, nor did the close proximity of two active sites increase the decay-accelerating activity. No decay of classical pathway C3-convertase was observed when dimeric CAR-Fc (Coxackie adenovirus receptor) or monomeric CAR were tested, confirming that the decay-accelerating activity measured was specific to KCP (Fig. 2).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   Acceleration of decay of classical C3-convertase for Fc fusion proteins (A) or released monomers (B). EAC142 were incubated in DGVB2+ alone or with varying concentrations of inhibitors for 5 min. The remaining active C3-convertases were measured by hemolysis after addition of guinea pig serum diluted in EDTA-containing buffer. Data are given as mean ± S.D. of quadruplicates.

KCP Shows Limited Inhibition of the Alternative Pathway C3-convertase-- The decay of the alternative pathway C3-convertase was also assessed by hemolytic assay. The alternative C3-convertase was assembled on the surface of sheep erythrocytes coincident with the addition of putative and known inhibitors, and decay acceleration was measured after 30 min. All three forms of KCP-Fc accelerated the decay equivalently, but they were 1000-fold less efficient compared with positive control DAF-Fc (Fig. 3A). Monomeric KCP proteins were also tested after being released by papain cleavage, but the decay-accelerating activity was not increased compared with dimeric Fc fusion proteins, and activity was found to be 1000-fold lower than the positive control FH (Fig. 3B). While KCP had poor alternative pathway decay-accelerating activity, it was still much greater than the negative control CAR-Fc and monomeric CAR, which had no significant decay-accelerating activity (Fig. 3).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Acceleration of decay of alternative C3-convertase for Fc fusion proteins (A) or released monomers (B). Increasing concentrations of inhibitors were added to EAC43 together with properdin, FD, and FB. The remaining active C3-convertases were measured by measuring released hemoglobin after the addition of guinea pig serum diluted in EDTA-containing buffer. Data are given as mean ± S.D. of quadruplicates.

KCP Acts as FI Cofactor in Cleavage of C4b-- Cofactor activity of KCP was assessed in a C4b-degradation assay in the presence of FI (Fig. 4, left panel). FI degradation of radiolabeled C4b was assessed in the presence or absence of putative or control cofactors under physiological saline conditions. All three forms of KCP-Fc were found to catalyze FI cleavage of the C4b alpha -chain from 85 kDa to the major fragment of 46 kDa, consistent with the cleavage of C4b to C4d (Fig. 5). Cleavage of C4b was not seen if C4b was incubated with KCP alone or FI alone, confirming co-operative activity. Further, no C4b cleavage was observed following incubation with DAF-Fc and FI (not shown), indicating the specificity for the KCP portion of the KCP-Fc molecule. All three forms were roughly equivalent in their ability to act as FI-cofactors, and 50% of the total C4b was converted to C4d by FI when KCP was present at 1.85 µM, 2.7 µM, and 1.55 µM, respectively, for short, medium, and long KCP-Fc forms compared with C4BP, which had a 50% total cofactor activity at 0.2 µM (not shown). However, this quantitative difference does not take into account that C4BP has seven active sites compared with the two active sites of KCP-Fc. Stably transfected CHO cells expressing GPI-anchored KCP short, medium, or long forms or unmodified full-length KCP, were also able to act as FI cofactors for the cleavage of C4b to C4d, while control CHO cells were unable to support FI activity (Fig. 5A). Hence, KCP was capable of FI cofactor activity in both cell-bound and soluble forms.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Diagrammatic depiction of the FI cleavage sites for C3b and C4b in the presence of various cofactors.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 5.   C4b-degradation assay for CHO cells expressing GPI-anchored monomeric forms of short (S), medium (M), or long (L) and CHO cells expressing naturally occurring transmembrane form (TM) (A) or forms of KCP-Fc (S, M, L) were examined (B). Positive control source of CR1 were human erythrocyte ghosts (A) or C4BP (B). Samples containing 125I-labeled C4b, FI, and indicated cofactors were incubated at 37 °C for 2 h. They were then mixed with a sample buffer containing reducing agent and heated, and the proteins were separated by 10-15% gradient SDS/PAGE. Radioactive C4b fragments were then visualized by autoradiography.

KCP Displays Novel FI Cofactor Activity in Cleavage of C3b-- All three forms of KCP-Fc were also tested for FI cofactor cleavage of C3b (C3b degradation products depicted in Fig. 4, right panel) as detailed above for C4b studies. FH and all three forms of KCP-Fc, but not DAF-Fc, were capable of inducing FI cleavage of the 104-kDa C3b alpha -chain releasing a product of 64 kDa as well as a 40-38-kDa doublet consistent with the formation of iC3b (Fig. 6). Moreover, all three KCP-Fc forms, but not FH, induced further cleavage of iC3b to a 30-kDa degradation product that was smaller than the 35-kDa C3dg degradation product produced by FI in the presence of CR1 (Fig. 6A). N-terminal sequencing of the 30-kDa species yielded the sequence HLIVTPSGCGEQNMIGMTPT. This sequence confirmed that the first 47 amino acid residues of C3dg were missing, which is consistent with the molecular mass and sequence of C3d. Fifty percent of the C3b was degraded by FI to smaller fragments when KCP-Fc was present at concentrations of 1.9 µM, 2.15 µM, and 1.25 µM, respectively, for short, medium, and long isoforms. This level of activity was calculated relative to FH, which catalyzed 50% FI cleavage of total C3b to the 64- and 40-38-kDa cleavage products (as FH did not induce production of C3dg) when present at a concentration of 50 nM. No cleavage was observed in the absence of FI (Fig. 6B), and incubation of C3b with KCP alone did not result in any cleavage (not shown). All the cell-associated forms of KCP, including the naturally occurring form, were also found to be as effective as soluble KCP for the cleavage of C3b to iC3b and C3dg, while control CHO cells did not act as FI cofactors (Fig. 6C).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   C3b-degradation assay for short form of KCP-Fc and CR1 in the presence and absence of FI under non reducing conditions (A), soluble short (S), medium (M), and long (L) KCP-Fc forms under reducing conditions (B), and CHO cells expressing naturally occurring transmembrane (TM) or GPI-anchored S, M, L forms of KCP (C). Human erythrocyte ghosts were used as a source of CR1 used as a positive control (A) or FH (B), whereas untransfected CHO cells were included as a negative control (C). Cofactors were incubated with 125I-labeled C3b and FI for 1.5 h at 37 °C. Radioactive C3b fragments were visualized by autoradiography after separation by 10-15% gradient SDS/PAGE.

The ability of KCP to degrade cell-bound C3b was also measured by flow cytometry analysis. Sheep erythrocytes were coated with C3b in the absence of sensitizing antibody (EC3 cells) through three consecutive rounds of incubation with C3, FB, FD, and properdin under alternative pathway activating conditions. EC3 cells were then incubated overnight with FI alone or in the presence of various cofactor proteins, and the degree of C3b cleavage attained was measured by flow cytometry using a panel of C3d- and C3c-specific antibodies (Table I). The same amount of cell-surface C3d was detected irrespective of enzyme or cofactor added. However, C3c epitopes were almost entirely removed from cells incubated with FI in the presence of CR1 and KCP but not FH. The amount of C3c remained the same after overnight treatment with either FI alone or with buffer alone, indicating that no significant nonspecific cleavage of C3b occurred (Table I). The same trend in specific decreased fluorescence for cells incubated with KCP or CR1 was observed with two separate polyclonal antibodies against C3c as well as a polyclonal antibody against intact C3.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Flow cytometry analysis of sheep erythrocyte-bound C3b degradation products

Binding of C3b and C4b to KCP Proteins Assessed by Surface Plasmon Resonance (Biacore)-- The affinity of binding between the KCP proteins and C3b/C4b was studied with a Biacore 2000. KCP proteins were immobilized on the surface of a CM5 chip using amino coupling. The three different constructs occupied different flow cells of the chip. C3b and C4b were then injected for sufficient time to reach saturation (Fig. 7A), and then KD was calculated from a binding curve showing response at equilibrium (Req) plotted against concentration (steady state affinity model; Fig. 7B). Binding of C3b/C4b to all three forms of KCP was evaluated both in dimeric form as well as papain-released monomeric form (Table II). Buffers used contained either 75 or 150 mM NaCl, and 2.5 mM CaCl2, and in some buffers 20 µM ZnCl2 was included (Table II). The reason for testing these two ions is that our preliminary homology-based three-dimensional model of CCP domains of KCP predicts binding sites for both calcium and zinc. The results obtained clearly show that all three KCP species are capable of binding C3b and C4b. In most conditions tested the affinity of KCPs for C4b was at least 10-fold higher than the affinity for C3b. Conversion of dimeric Fc fusion protein to monomers was necessary to accurately measure binding to C3b and C4b under physiological salt (150 mM) conditions, possibly due to the removal of steric hindrances. The addition of physiological levels of zinc (20 µM) increased the binding of KCP to C4b 10-fold under all conditions but was less efficient at increasing KCP binding to C3b. Addition of calcium to buffers did not affect the affinity constants (not shown).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Surface plasmon resonance analysis. A, purified C4met was injected at various concentrations over a CM5-chip with short, medium, and long forms of KCP immobilized in separate flow cells. Identical samples were injected over a control flow cell, and nonspecific binding was subtracted. The amount of C4met associated with KCP was measured in arbitrary resonance units. B, the response obtained for each concentration of C4met at equilibrium (Req) was plotted against concentration, which allowed for estimation of KD.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Summary of surface plasmon resonance analysis (equilibrium dissociation constants)


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Virus-encoded proteins with homologies to host complement regulators have been previously described in two families: Herpesviridae and Poxviridae. Vaccinia complement control protein (VCP) was the first described and is the most completely characterized (21-26). Other poxviruses that retain homologues to VCP in their genome include variola virus (small pox), cowpox, and monkeypox, the latter missing the fourth CCP domain and perhaps not functional (27-30). With the exception of Herpes Simplex virus type 1, which encodes glycoprotein C, a complement regulator that does not have homology to any known inhibitor, all herpesviruses that encode complement regulators belong to the rhadinovirus genus. Such members include HVS, Mgamma HV68, and KSHV. The genomic organization has been conserved between the different rhadinoviruses so that the gene encoding the complement regulator is designated as ORF4 (5, 10, 31-33).

Although the functional complement-regulating ability of all the above listed proteins has been studied, there are no reports testing decay-accelerating activity separately from FI cofactor activity as we have detailed here for KSHV. We used a FI-deficient purified protein system, and we showed that KCP has true decay-accelerating activity for the classical pathway C3-convertase but is poor at enhancing the decay of alternative pathway C3-convertase. The ability of KCP to decay classical C3-convertase is 10-fold lower than for DAF-Fc but only 5-fold less than that observed for C4BP. However, it is important to note that C4BP is a heptameter with several equivalent binding sites and therefore activity of the single active site in KCP is comparable to that of C4BP. Moreover, the local concentration of KCP in vivo produced from KSHV-infected cells undergoing lytic replication would be high at the site of complement activation (i.e. the virally infected cell) and therefore would be far more effective than soluble host complement regulators anyway. Since KCP seems to be fairly specific for the regulation of the classical pathway as its ability to decay alternative C3-convertase was not very strong (1000-fold lower than DAF-Fc and FH), it suggests that the alternative pathway may not play a significant role in clearance of the virus in vivo. This is in contrast to the homologous viral complement regulator encoded by Mgamma HV68, which has been reported to inhibit both alternative and classical pathways of whole mouse and human serum (11).

We determined that KCP has FI cofactor activity for the cleavage of C4b and C3b. FI cofactor activity has not been tested for any of the other rhadinoviruses, but VCP and the variola virus-encoded complement regulator SPICE have both been shown to act as FI cofactors in the cleavage of C4b and C3b (23, 24, 29). Interestingly, SPICE but not VCP has been suggested to mediate FI cleavage to the C3dg fragment, previously only described for CR1 (29, 34). However, SPICE and KCP cofactor activities are distinctly different in that the final FI degradation product for C3b in the presence of KCP was C3d not C3dg as was reported for SPICE (Fig. 6 and Ref. 29), which we confirmed through N-terminal sequence of the isolated degradation product. FI has never been shown to cleave at this site before, even in the presence of CR1, and the C3d fragment has only been reported as a cleavage product of trypsin, plasmin, or elastase (35). The most common FI cleavage sites in C3b are the two Arg-Ser sequences that result in the release of C3f; however, in the presence of CR1, FI cleaves iC3b at an Arg-Glu sequence and under non-physiological conditions has been reported to cleave iC3b at a Lys-Glu sequence (36). It is therefore surprising that under physiological saline conditions we observed a FI cleavage of C3b at a Glu-His sequence in the presence of KCP. The mechanism by which this occurs is under further investigation. It is important to point out that KCP is the only viral protein that has been shown to degrade cell-associated C3b to C3d, proving that this activity is not an artifact of soluble phase experiments (Table I).

We measured the binding affinity of KCP to C4b and C3b (Table II) using Biacore. These data indicate that all three KCP species are capable of binding C3b and C4b but that the affinity of KCP for C4b is at least 10-fold higher than for C3b. The addition of physiological levels of zinc increased the binding of KCP to C4b 10-fold under all conditions. The higher binding affinity of KCP for C4b, relative to C3b binding, also supports our findings that KCP is better at accelerating the decay of the classical, compared with the alternative, C3-convertase. In comparison to host-encoded complement regulators, we found the that the affinity of KCP for C4b was 7-fold less than that measured for C4BP (KD = 0.5 µM in 150 mM NaCl and 5 mM EDTA), and for C3b was 20-fold less than FH (KD = 0.4 µM in 150 mM NaCl and 5 mM EDTA) under the same conditions (not shown).

The ability of KCP to catalyze the cleavage of C3b to C3d by FI is unique among the virus and host complement regulators. It will be important to study the FI cofactor activity for the other rhadinovirus members HVS and Mgamma HV68, as these have not yet been investigated. There may be an evolutionary reason for KSHV or KSHV-infected cells to promote the production of C3d: the receptor for C3d is CR2, expressed predominantly by B-cells, which has been identified as one of the KSHV viral reservoirs (37, 38). Thus KSHV may have evolved a mechanism that not only allows evasion of clearance of virions and virus-infected cells but may be using the complement system to target the virus to primary sites of pathogenesis during natural infection as B-cell infection by KSHV is a central aspect of primary effusion lymphoma and Castleman's disease (reviewed in Ref. 3 and 4). However, KCP is only expressed in infected cells during lytic replication in B cells (9). While this expression profile may facilitate KSHV transmission from infected cells to uninfected B-cells, whether KCP facilitates infection by extracellular virions through expression on the envelope is currently under investigation.

The importance of complement evasion by rhadinoviruses has recently been underscored in a thorough pathological examination of Mgamma HV68 infection compared with a recombinant virus lacking the complement regulating homologue (39). Deletion of ORF4 from Mgamma HV68 resulted in a dramatic decrease in virulence during acute central nervous system infection of immunocompetent mice and in mortality in persistently infected immunocompromised mice, and this attenuation was not seen in C3-deficient mice. Furthermore, C3 played a role in regulating latency of the virus, but the Mgamma HV68 ORF4 could not counteract this effect (39). This also emphasizes the importance of KCP inhibition of the classical pathway C3-convertase and degradation of cell-bound C3b and C4b that we have detailed here. Extended to in vivo infections, these complement-regulating activities would facilitate KSHV replication and enhance virus pathogenesis through a combination of blocking virion and/or infected cell opsonization, blocking complement-mediated virolysis, and inhibiting anaphylaxis.

    ACKNOWLEDGEMENTS

We thank Dr. Kevin Marchbank (University of Wales College of Medicine, Cardiff, UK) for guidance and discussion on C3dg studies, Profs. Patrick Moore and Yuan Chang (University of Pittsburgh Cancer Institute, Pittsburgh, PA) for valuable provision of resources and discussion, Dr. Bruno Villoutreix (University of Paris) for homology-based modeling, and Prof. B. Paul Morgan (University of Wales College of Medicine) and Prof. Björn Dahlbäck (Lund University, Malmö, Sweden) for continuous generous support.

    FOOTNOTES

* This study was supported by grants from the Swedish Research Council (to A. B.), Kock's Trust, Österlunds Trust, Crafoord Trust, Royal Physiographic Society in Lund, Groschinsky's Trust, King Gustav V's 80th Anniversary Foundation, Zoega's Trust, and University Hospital in Malmö. This study was also supported by the Wellcome Trust Foundation, Cancer Research UK (to O. B. S. and D. J. B.) Grant C7934 and the Association for International Cancer Research Grant 01-242 (to D. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Wellcome Trust Career Development Fellow.

** To whom correspondence should be addressed. Tel.: 46-40-33-82-33; Fax: 46-40-33-70-44; E-mail: Anna.Blom@klkemi.mas.lu.se.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211579200

    ABBREVIATIONS

The abbreviations used are: KSHV, Kaposi's sarcoma-associated virus; HVS, Herpesvirus saimiri; ORF, open reading frame; DAF, decay accelerating factor; CCP, complement control protein; CHO, Chinese hamster ovary; FB, factor B; FD, factor D; FH, factor H; FI, factor I; C4BP, C4-binding protein; GPI, glycosylphosphatidyl inositol; KCP, KSHV complement control protein; FCM, flow cytometry medium; VCP, vaccinia complement control protein; CAR, Coxackie adenovirus receptor; CR1, complement receptor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M., and Moore, P. S. (1994) Science 266, 1865-1869[Medline] [Order article via Infotrieve]
2. Antman, K., and Chang, Y. (2000) N. Engl. J. Med. 342, 1027-1038[Free Full Text]
3. Cesarman, E., and Knowles, D. M. (1999) Semin. Cancer Biol. 9, 165-174[CrossRef][Medline] [Order article via Infotrieve]
4. Schulz, T. F. (2001) Eur. J. Cancer 37, 1217-1226[CrossRef][Medline] [Order article via Infotrieve]
5. Russo, J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M., Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y., and Moore, P. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14862-14867[Abstract/Free Full Text]
6. Jenner, R. G., and Boshoff, C. (2002) Biochim. Biophys. Acta 1602, 1-22[CrossRef][Medline] [Order article via Infotrieve]
7. Norman, D. G., Barlow, P. N., Baron, M., Day, A. J., Sim, R. B., and Campbell, I. D. (1991) J. Mol. Biol. 219, 717-725[Medline] [Order article via Infotrieve]
8. Kirkitadze, M. D., and Barlow, P. N. (2001) Immunol. Rev. 180, 146-161[CrossRef][Medline] [Order article via Infotrieve]
9. Spiller, B. O., Robinson, M., O'Donnell, E., Milligan, S., Morgan, P. B., Davison, A. J., and Blackbourn, D. J. (2003) J. Virol. 77, 592-599[CrossRef][Medline] [Order article via Infotrieve]
10. Albrecht, J. C., and Fleckenstein, B. (1992) J. Virol. 66, 3937-3940[Abstract]
11. H. W.Kapadia, S. B., Molina, H., van Berkel, V., Speck, S. H., and Virgin, H. W., IV (1999) J. Virol. 73, 7658-7670[Abstract/Free Full Text]
12. Dahlbäck, B. (1983) Biochem. J. 209, 847-856[Medline] [Order article via Infotrieve]
13. Gigli, I., Porter, R. R., and Sim, R. B. (1976) Biochem. J. 157, 541-548[Medline] [Order article via Infotrieve]
14. Dahlbäck, B., and Hildebrand, B. (1983) Biochem. J. 209, 857-863[Medline] [Order article via Infotrieve]
15. Andersson, M., Hanson, A., Englund, G., and Dahlbäck, B. (1991) Eur. J. Clin. Pharmacol. 40, 261-265[Medline] [Order article via Infotrieve]
16. Blom, A. M., Kask, L., and Dahlbäck, B. (2003) Mol. Immunol. 39, 547-556[CrossRef][Medline] [Order article via Infotrieve]
17. Crossley, L., and Porter, R. (1980) Biochem. J. 191, 173-182[Medline] [Order article via Infotrieve]
18. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123
19. Harris, C. L., Spiller, O. B., and Morgan, B. P. (2000) Immunology 100, 462-470[CrossRef][Medline] [Order article via Infotrieve]
20. Zabern, I., Bloom, E., Chu, V., and Gigli, I. (1982) J. Immunol. 128, 1433-1438[Abstract/Free Full Text]
21. Kotwal, G. J., and Moss, B. (1988) Nature 335, 176-178[CrossRef][Medline] [Order article via Infotrieve]
22. Kotwal, G. J., Isaacs, S. N., McKenzie, R., Frank, M. M., and Moss, B. (1990) Science 250, 827-830[Medline] [Order article via Infotrieve]
23. McKenzie, R., Kotwal, G. J., Moss, B., Hammer, C. H., and Frank, M. M. (1992) J. Infect. Dis. 166, 1245-1250[Medline] [Order article via Infotrieve]
24. Sahu, A., Isaacs, S. N., Soulika, A. M., and Lambris, J. D. (1998) J. Immunol. 160, 5596-5604[Abstract/Free Full Text]
25. Rosengard, A. M., Alonso, L. C., Korb, L. C., Baldwin, W. M., 3rd, Sanfilippo, F., Turka, L. A., and Ahearn, J. M. (1999) Mol. Immunol. 36, 685-697[CrossRef][Medline] [Order article via Infotrieve]
26. Murthy, K. H., Smith, S. A., Ganesh, V. K., Judge, K. W., Mullin, N., Barlow, P. N., Ogata, C. M., and Kotwal, G. J. (2001) Cell 104, 301-311[Medline] [Order article via Infotrieve]
27. Miller, C. G., Shchelkunov, S. N., and Kotwal, G. J. (1997) Virology 229, 126-133[CrossRef][Medline] [Order article via Infotrieve]
28. Kotwal, G. J., Miller, C. G., and Justus, D. E. (1998) Mol. Cell Biochem. 185, 39-46[CrossRef][Medline] [Order article via Infotrieve]
29. Rosengard, A. M., Liu, Y., Nie, Z., and Jimenez, R. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8808-8813[Abstract/Free Full Text]
30. Uvarova, E. A., and Shchelkunov, S. N. (2001) Virus Res. 81, 39-45[CrossRef][Medline] [Order article via Infotrieve]
31. Virgin, H. W., IV, Latreille, P., Wamsley, P., Hallsworth, K., Weck, K. E., Dal Canto, A. J., and Speck, S. H. (1997) J. Virol. 71, 5894-5904[Abstract]
32. Fodor, W. L., Rollins, S. A., Bianco-Caron, S., Rother, R. P., Guilmette, E. R., Burton, W. V., Albrecht, J. C., Fleckenstein, B., and Squinto, S. P. (1995) J. Virol. 69, 3889-3892[Abstract]
33. Rother, R. P., Rollins, S. A., Fodor, W. L., Albrecht, J. C., Setter, E., Fleckenstein, B., and Squinto, S. P. (1994) J. Virol. 68, 730-737[Abstract]
34. Medof, M. E., and Nussenzweig, V. (1984) J. Exp. Med. 159, 1669-1685[Abstract]
35. Dodds, A. W., and Sim, R., B. (1997) in The Practical Approach Series (Rickwood, D. , and Hames, B. D., eds) , p. 182, IRL Press, Oxford
36. Ekdahl, K. N., Nilsson, U. R., and Nilsson, B. (1990) J. Immunol. 144, 4269-4274[Abstract/Free Full Text]
37. Ambroziak, J. A., Blackbourn, D. J., Herndier, B. G., Glogau, R. G., Gullett, J. H., McDonald, A. R., Lennette, E. T., and Levy, J. A. (1995) Science 268, 582-583[Medline] [Order article via Infotrieve]
38. Mesri, E. A., Cesarman, E., Arvanitakis, L., Rafii, S., Moore, M. A., Posnett, D. N., Knowles, D. M., and Asch, A. S. (1996) J. Exp. Med. 183, 2385-2390[Abstract]
39. Kapadia, S. B., Levine, B., Speck, S. H., and Virgin, H. W. (2002) Immunity 17, 143-155[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.