1245
Regulation of Complement Activity by Vaccinia Virus Complement-Control Protein Robin McKenzie, Girish J. Kotwal,* Bernard Moss, C. H. Hammer,* and M. M. Frank*
Laboratories ofClinical Investigation and Viral Diseases. National Institute ofAllergy and Infectious Diseases. National Institutes ofHealth. Bethesda. Maryland
The complement system is well defined as one of the principal host mechanisms defending against a wide variety of microorganisms. Most studies have centered on the role of complement in opsonization and lysis of bacteria, but complement attacks a variety of other microorganisms, including some viruses. Recently, investigators have discovered that some viruses counter this attack by producing proteins that inhibit complement activation [1-7]. Human blood contains multiple complement-regulatory proteins. These factors control activation of this crucial defense system and regulate generation of tissue-damaging protein fragments and complexes. In one large complementregulatory family, the individual proteins are made up of 4-30 short consensus repeats, each ofwhich is 60-70 amino acids long. C4-binding protein is spider-shaped, with seven 70-kDa tentacles radiating out from a central core [8]. Each tentacle contains 8 short consensus repeats. This protein binds the activated fourth component of complement, C4b, and regulates the classical complement cascade at several steps. Vaccinia virus-infected cells secrete in abundance a 35kDa protein that is structurally similar to C4-binding protein and other complement-control proteins [9]. The four short consensus repeat sequences of vaccinia complement-control protein (VCP) show marked homology to those in C4-binding protein (38%). VCP inhibits hemolysis ofsensitized sheep erythrocytes in human serum by interfering with cornple-
Received 24 January 1992; revised 22 July 1992. Reprints or correspondence: Dr. Robin McKenzie, Laboratory of Clinical Investigation, NIAID, Bldg. 10. Room l1N228. National Institutes of Health. Bethesda, MD 20892. * Present affiliations: James N. Gamble Institute of Medical Research. Cincinnati. (G.J.K.): FCRDCjNIAID. Fort Detrick, Frederick, Maryland (C.H.H.); Department of Pediatrics. Duke University Medical Center, Durham, North Carolina (M.M.F.). The Journal of Infectious Diseases 1992;166:1245-50 This article is in the public domain.
ment-mediated attack [10]. Animal studies have shown decreased virulence of a mutant virus that lacks the gene encoding VCP [II]. We investigated the mechanisms by which VCP inhibits complement activity, thereby increasing the pathogenicity of vaccinia virus.
Materials and Methods Buffirs and reagents. The following buffers were used: isotonic veronal buffered saline with 0.1%gelatin (GVBS); GVBS with 0.15 mM calcium chloride and 1.0 mM magnesium chloride (GVBS++); GVBS++ diluted 60:40 with 5% dextrose containing 0.1% gelatin, 0.15 mM calcium chloride, and 1.0 mM magnesium chloride (DGVBS++); GVBS with 20 mM EDT A (EDTA-GVBS); GVBS with 10 mM EGTA and 2 mM magnesium chloride (Mg/EGTA); Mg/EGTA diluted 60:40 with 5% dextrose containing 0.1% gelatin, 10 mM EGT A, and 2 mM magnesium chloride (60% mg/EGTA buffer); PBS; and Alsever's solution (0.11 M dextrose, 27 mM sodium citrate, 72 mM sodium chloride). Sources ofcomplement components. Human C4 and C3 were purified by the method of Hammer et al. [12] modified to include passage over a fast protein liquid chromatography anion exchange column (Fast Q; Pharmacia, Piscataway, NJ) [13]. Final concentrations of C4 and C3 were 70 and 500 JLg/mL, with activities of 40,000 and 50,000 units/ml., respectively. One unit was defined as the reciprocal of the dilution required to produce 63% lysis of antibody-coated sheep red blood cells at 1.5 X 108 cells/rnl. [14]. Purity assessed by Coomassie blue staining of7.5% SDS-polyacrylamide gels was >95%. To cleave C4 and produce C4b, fractions from the Fast Q column shown to contain C I protease activity were incubated at 37°C overnight. Paranitrophenyl guanidinobenzoate (Sigma, St. Louis) and aprotinin (Sigma) were then added before storage at -85°C. C3 was converted to C3b by incubation with varying concentrations of trypsin for 3 min at 37°C [15]. Proteolysis from these two methods was then assessed by SDS-PAGE. Guinea pig C I and human C2 were purchased from Diamedix (Miami). Serum from guinea pigs genetically deficient in C4
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A major protein secreted by vaccinia virus-infected cells has structural similarity to the superfamily of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.
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McKenzie et al.
Richmond, CA) using a NaCl gradient followed by passage through Sephadex G-100 (Pharmacia) with GVBS++ as the buffer [10]. The protein concentration ofthe final eluant was 67 ~g/mL as determined by bicinchoninic acid assay (Pierce, Rockford, IL). Purity was assessed by SDS-PAGE. Radiolabeling. Purified C3, purified C4, and concentrated medium (300X) from vaccinia virus-infected cells were radiolabeled with 1251 using lodobeads (Pierce) according to the manufacturer's instructions. The labeled proteins were separated from free iodine using a PD-I 0 column (Pharmacia) eluted with PBS. Specific activities of C3 and C4 were 2.2 X 106 and 1.3 X 106 cpm/ug, respectively. Five percent bovine serum albumin was added to the purified proteins before storage at -70°C. SDS-PAGE. SDS-PAGE was done on 7.5% and 12% gels under reducing conditions [20]. Autoradiograms were analyzed by densitometry using an Ultroscan XL laser densitometer (LKB, Piscataway, NJ). Binding to C4b- and C3b-Sepharose. C4b, C3b, and human serum albumin were coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. Protein (5-10 mg) was coupled to each milliliter of beads. 1251-labeled medium from vaccinia virus-infected cells was applied to columns of C4b- or C3b-Sepharose. After thorough washing with DGVBS++, columns were eluted with DGVBS++ containing I M NaCI. Eluants were desalted on PD10 columns (Pharmacia) using PBS as the buffer. Lysis ofEAC14b in C4D serum. Since C4D serum provides all complement components except C4, EACI4b will lyse in C4D serum. To determine whether VCP would block this lysis, EAC 14b ( 1.5 X 108 /mL, made with about one active hemolytic C4b site per cell) were incubated for 30 min at 37°C with either medium from cells infected with vaccinia virus, medium from cells infected with the 6/2 mutant, or purified VCP. Then C4D serum diluted 1:75 in DGVBS++ was added. After 60 min, lysis of erythrocytes was determined by reading the optical density at 412 nm. Effect on classical C3 convertaseformation. C4-binding protein inhibits the formation of the classical C3 convertase. Experiments were, therefore, done to examine the effect of media from vaccinia virus-infected cells on the formation of C 142a, the classical C3 convertase. Increasing concentrations ofC2 in a volume of I00 ~L were added to tubes containing I00 ~L of either DGVBS++ buffer, a IX or Y3X concentration of medium from vaccinia virus-infected cells, or a IX concentration of medium from cells infected with the vSIGK3 mutant virus. EAC14b (100 ~L at 1.5 X 108/mL, made with limited C4) were added. Tubes were incubated for 4 min at 30°C. ComplementEDT A (I mL) was then added, and incubation continued for an additional 60 min at 37°C. After centrifugation, lysis was quantitated by measuring the optical density of the supernatants at 412 nm. Results were expressed as Z, the number of hemolytically active sites per erythrocyte. Effect on classical C3 convertase decay. C4-binding protein accelerates the decay of EACI42a. Experiments were done to determine if VCP affects this rate of decay. EAC 14b (I 00 ~L at 1.5 X 108/mL, made with about one hemolytically active C4b site per cell) were incubated with I00 ~L ofC2, 20 units/rnl., for 4 min at 30°C. Cells were washed with ice-cold DGVBS++ and
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(C4D serum) obtained from our own colony of guinea pigs [16] contained no C4 by hemolytic assay. Complement-EDTA, used as a source of C3-C9, was prepared by incubation of normal guinea pig serum in EDTA-GVBS at a I:25 dilution for 15 min at 3rc. (EDTA chelates calcium and magnesium needed for activation of Cl , C4, and C2 but does not interfere with activation ofC3-C9.) Factors I, B, and D were purchased from Quidel (San Diego). Factor H was purified as reported [17]. Cellular intermediates. Complement can be activated by the classical and alternative pathways. Each of these pathways produces a C3 convertase that activates C3 and then C5-C9. Activation of the complement components through C9 on the surface of a sheep erythrocyte causes lysis of the erythrocyte. C142a, the classical C3 convertase, and C43Bb, the alternative pathway C3 convertase, were prepared as follows. Sensitized sheep erythrocytes (EA) were used as targets to activate and bind complement components. EA were sequentially incubated with purified C I followed by C4 to produce EACI4b and further incubated with C2 to produce EACI42a [18]. EAC 14b with limited C4 sites were prepared by incubating EAC I (1.5 X 108 /mL) in DGVBS++ containing I unit/rnl. purified C4. EAC43b were made by incubating EACI (5 X 108/mL) first with C4, 33 units/ml., for 45 min at 37°C and then with C2, 300 units/rnl., and C3, 450 units/ml., for 30 min at 37°C. Cells were then incubated in 10 mM EDT A for two 20-min incubations at 37°C. The resulting EAC43b did not lyse when 0.1 mL of cells was incubated for I h at 37°C in I mL of complementEDTA, showing the absence ofC3 convertase activity. EAC43Bb were formed by incubating EAC43b (2.4 ml., 1.5 X 108 cells/ml.) with varying amounts of factor B and factor D for 30 min at 30°C. To calculate the number of hemolytically active sites per cell, 0.1 mL ofEAC43Bb was added to I mL of complement-EDTA and the mixture incubated for I h at 37°C. After centrifugation, the optical density of the supernatants was read at 412 nm and the percentage oflysis (y) determined. The average number of hemolytically active sites per cell (2) was then determined according to the formula: Z = -In(1 - y) [14]. Incubation of EAC43b with 60 ng of factor Band 400 ng of factor D produced EAC43Bb with about one active site per cell. Viral culture and purification of vCP. RK 13 cells (CCL 37; American Type Culture Collection, [ATCC], Rockville, MD) in serum-free OPTI-MEM I medium (GlBCO, Grand Island, NY) were infected with 5 pfu/cell of either wild-type vaccinia virus (strain WR, VR1354; ATCC) or one of two mutants: 6/2, a spontaneous mutant lacking a cluster ofgenes including the one that encodes VCP [19], or vSIGK3, a recombinant mutant with a 70-bp segment within the VCP gene replaced by the selectable marker gene, xanthine-guanine phosphoribosyltransferase, regulated by a vaccinia promoter [10]. After a l-h incubation, the monolayer was washed with serum-free OPTI-MEM and reincubated in this medium for 24 h. Tissue culture medium from infected cells was then centrifuged at 2000 g for 10 min, concentrated 25x-300X using a Centricon 30 (Amicon, Danvers, MA), and diluted in DGVBS++ before use. Concentrations are expressed relative to unconcentrated, undiluted tissue culture medium. VCP was isolated from concentrated medium by two chromatographic procedures: elution from DEAE-Biogel (Bio-Rad,
JID 1992;166 (December)
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Vaccinia Complement-Control Protein
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Results Figure 1. Binding of VCP to C4b- and C3b-Sepharose. SDSPAGE and autoradiography of (2SI-labeled tissue culture me- 20093dium (lanes I, 5), column washes 69(lanes 2,3,6,7), and desalted col- 46umn eluants (lanes 4,8) show 35kDa bands in high-salt eluents 30but not washes. 14-
100
80
"U
60
e
~
0 Gl
.,.
40
20
0 0
20
40
60
80
100
Time (min)
Figure 2. Binding of purified 12sI-labeled VCP to C4b-Sepharose at timed intervals.
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resuspended in this buffer at 1.5 X 108/m L. The resulting EACI42a were allowed to decay at 30°C during incubation with buffer, with medium from vaccinia virus- or 6/2 mutant virus-infected cells, or with purified VCP at a final concentration of 1.3 or 0.67 ILg/mL. At timed intervals, 0.2 mL of the cell suspension was mixed with I mL ofcomplement-EDTA and the mixture incubated at 37°C for 60 min. After centrifugation, the optical density of the supernatant was measured at 412 nm. Effict on alternative pathway convertase decay. EAC43Bb, 0.1 mL, made as above were incubated for 6 min at 30°C with 0.1 mL ofDGVBS++ or VCP at concentrations indicated. Complement-EDTA (I mL) was added and the mixture incubated an additional 60 min at 3rc. The optical density at 412 nm was then measured. Regeneration of classical C3 convertase activity after decay. To determine whether the classical C3 convertase was reversibly or irreversibly altered during decay with VCP, C2 was added back to the convertase after decay. The C42a convertase activity ofEACI42a (1.5 X 108/mL) prepared with limited C4 was allowed to decay in the presence of DGVBS++ buffer or medium from vaccinia-infected cells (3X final concentration). As a control, EACI4b were incubated with buffer. After 30 min, cells were washed twice in cold DGVBS++,and remaining C42a sites were quantitated as above by incubating 200 ILL of each cell suspension with I mL of complement-EDTA and measuring lysis of cells. To regenerate decayed C42a sites, another aliquot of the cell suspension after decay was mixed with C2 at 50 units/ mL. After incubation at 30°C for 4 min, cells were washed and added to complement-EDTA to quantitate hemolytically active C42a sites. Cofactor activity for the cleavage of C4b and C3b by factor I. In the presence of a cofactor such as factor H or C4-binding protein, factor I cleaves C3b and C4b. Experiments were done to see if VCP has similar cofactor activity. EACI4b and EACI423b prepared with 12sI-labeled C4 and 12sl-labeled C3, respectively, were incubated for 60 min at 37°C with factor I, VCP, or factor H and with combinations of these factors at the concentrations indicated. Cells were then washed in DGVBS++. EACI423b were incubated for I h at 37°C in 0.025 M methylamine buffer containing 0.05 M sodium bicarbonate, 1% SDS, and 25 ILM paranitrophenyl guanidinobenzoate (Sigma) to free ester-bound radiolabe1ed C3 from the erythrocyte. After neutralization with 0.1 N HCI, these samples and the EAC 14b were boiled in SDS buffer containing 2-mercaptoethanol and analyzed by SDS-PAGE.
Binding to C4b- and C3b-Sepharose. Medium from cells infected with wild-type vaccinia virus was concentrated, labeled with 1251, and applied to columns packed with C4b- or C3b-Sepharose. Columns were washed with DGYBS++ and then eluted with DGVBS++ containing 1 M saline. SDSPAGE of the high-salt eluants showed a predominant band with the expected molecular mass ofYCP, 35 kDa (figure 1). In a timed experiment, most binding to C4b-Sepharose occurred within 5 min, with no further increase in binding after 30 min (figure 2). To determine whether the material eluted from the C4b-Sepharose column would bind to C3b, the eluant was desalted and added to duplicate tubes containing either C4b-Sepharose beads, C3b-Sepharose beads, or human serum albumin-Sepharose beads. Tubes were rotated at 4°C for 30 min. Seventy-five percent of the 1251-labeled material was rebound by the C4b-Sepharose, 60% was bound by the C3b-Sepharose, and 13% by the control, human serum albumin-Sepharose. A decrease in rebinding to C4b-Sepharose may have been due to less efficient contact between YCP and C4b in rotating tubes or to some denaturation of the protein. The ability of most of the radioactive material eluted from the C4b column to rebind to C3bSepharose and the similarity in the bands on SDS-PAGE (figure 1) indicate that the same 35-kDa protein binds to both C3b and C4b. Inhibition of the lysis ofEAC14b in C4D serum. Lysis of EAC 14b in C4D serum is inhibited in a dose-response fashion by medium from cells infected with vaccinia virus but not by medium from cells infected with the 6/2 mutant [10]. In the current experiments, 50% inhibition occurred when vaccinia virus medium was present at a final dilution of I in
McKenzie et al.
1248
0.2-r--------------,
z
0.1
40
C2
60
80
'00
Buffer
--0--
vSIGK3, 1X
---0-
WR. 113X
____
WR,lX
120
(units/mil
Figure 3. InhibitionofC42a convertase formation on sensitized sheep erythrocytes. WR, medium made from cells infected with wild-type vaccinia virus; vSIGK3, medium fromcellsinfectedwith vSIGK3 mutant virus. Z. mean number of hemolytically active sites per erythrocyte.
4. Lysis was also inhibited by purified VCP. About 50%inhibition occurred when VCP was present at a final concentration of 4 ~g/mL. No inhibition was produced by medium from cells infected with the 6/2 mutant. To determine whether preincubation ofEACl4b with the vaccinia protein was necessary to produce inhibition, cells were incubated with medium from vaccinia virus-infected cells for 0-30 min at 37°C or O°C before the addition of C4D serum. No change in the percentage of inhibition occurred under these conditions. Inhibition ofclassical C3 convertaseformation. EACl4b with limited amounts of C4b were incubated with increasing amounts of C2 together with buffer, a 1X or Y3X concentration of medium from vaccinia virus-infected cells, or a 1X concentration of medium from vSIGK3 virus-infected cells. Classical pathway C3 convertase activity was then determined by further incubation with complementEDT A. Formation of the convertase was inhibited by medium from wild-type vaccinia virus but not by medium from the mutant virus at all concentrations of C2 tested (figure 3). An additional experiment was done to see whether interaction of VCP with EAC 14b before addition of C2 was required for the inhibitory effect. In one set of duplicate tubes, EAC 14b were incubated first with 1X vaccinia virus medium for 4 min, followed by the addition of excess C2 (10 units/ mL) for an additional 4 min of incubation. In a second set of tubes, EAC 14b were incubated first with C2 for 4 min before the addition of 1X medium for another 4 min. In a third set of tubes, EAC 14b were incubated with C2 and 1X medium together for 8 min. Under these three conditions, inhibition was 56%, 56%, and 54%, respectively, showing no difference. Acceleration of classical C3 convertase decay. EACl4b
made with about one hemolytically active site per cell were incubated with C2, 20 units/rnl., for 4 min at 30°C. The EACl42a were washed, then incubated with buffer or with medium from vaccinia virus- or 6/2 virus-infected cells. At timed intervals, 0.2 mL of the cell suspension was mixed with 1 mL of complement-EDTA, the mixture incubated at 37°C for 60 min, and lysis measured. When EAC 142a were incubated in buffer or medium from 6/2-infected cells diluted 1 in 2, the half-life of the C42a enzyme was 7 min. When medium from vaccinia virusinfected cells was present at a dilution of 1 in 6.7 or 1 in 2.5, the half-life decreased to 2 min and 1 min, respectively. Medium from cells infected with the 6/2 mutant produced no change in the decay rate. Purified VCP also accelerated the decay of the C42a convertase. When the protein was present at a final concentration of 0.67 ~g/mL or 1.3 ~g/mL, the half-life decreased to 4 min and 3 min, respectively (figure 4). Experiments were done to determine whether VCP destroyed the C4b site. After decay in the presence of either buffer or medium from vaccinia virus-infected cells, cells were washed in cold buffer. Convertase activity could then be completely restored by the addition offresh C2 (table 1). VCP, therefore, accelerates classical convertase decay by a reversible, nonproteolytic mechanism. Acceleration of alternative pathway convertase decay. Aliquots of EAC43Bb with a limited number of hemolytically active sites per cell were incubated for 6 min at 30°C with varying concentrations of purified VCP or DGVBS++. Complement-EDTA was then added and samples incubated at 37°C for 60 min. Hemolysis was quantitated by measuring the optical density at 412 nm. A control sample containing
10.,....
-,
z .1
0
BUFFER
&
VCP 0.67 UGIML
I
I
01
L~
o
10
20
vCP 1.3 UGIML
30
TIME (MIN)
Figure 4. Acceleration of C42a convertase decay on sensitized sheep erythrocytes by VCP.
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20
____
JID 1992; 166 (December)
Vaccinia Complement-Control Protein
110 1992; 166(December)
1249 2345678
Table 1. Reconstitution ofC42 convertaseafter decay in the presence of medium from vaccinia virus-infected cells. I: H:
C42 convertase activity (2)
Cells EACl42 EACl42 EACl4
Reagen t present during decay
After decay
After reconstitution withC2
Buffer 3x vaccinia medium Buffer
0.11 0 0
1.48 1.49 1.49
a{3-
a'{J-
y-
-
93 69
- +
3 4 5 6
+ + ....III
~
--~
Ql C
~
....III Ql
c
Figure 6. Cofactor activity for cleavage of C3b by factor I. Lane I, buffer;lane 2, factor I alone; lanes 3-5, factor I plus VCPat"0.67 ILg/mL, 6.7 lLg/mL, or 67 lLg/mL; lane 6, factor I plus factor H; lane 7, factor H alone; lane 8, VCP alone at 67 ILg/mL. Positions of 0/ and fl chains of nonactivated C3 standard are shown (120 kDa and 75 kDa, respectively). Molecular mass ofC3b 0/ chain is 110 kDa. Degradation of C3b 0/' chain produces 0/'1 (68 kDa) and 01'2 (43 kDa); O/'! chain is further degraded to 0/'3 (27 kDa) and 01'4 (41 kDa).
0.03ILg/mL (lane 2), or with factor I plus VCP at 0.67 ILg/mL (lane 3), 7% of the ex' chain was cleaved. Factor I, together with VCP at 6.7 ILg/mL (lane 4) or 67 ILg/mL (lane 5), cleaved 21 % and 57% of the 0/' chain, respectively. Factors I and H together (lane 6) cleaved 71 % of the 0/' band.
Discussion VCP, a major secretory protein of vaccinia virus, is structurally homologous to C4-bindtng protein [9]. Like C4-binding protein, VCP binds to C4b and regulates the classical complement pathway. This regulation controls the formation and decay ofthe classical C3 convertase and the degradation of C4b by factor I. These effects occur with concentrations ofVCP present in un concentrated, unpurified medium from vaccinia virus-infected cells. Similar to other complement regulatory proteins, VCP affects the alternative pathway. It binds to C3b and acts as a cofactor with factor I in the degradation of C3b. Although VCP accelerates the decay of the alternative pathway convertase, under the conditions examined higher concentrations were required than were needed to affect the classical pathway convertase. It is generally believed that cellular immunity is more prominent than humoral immunity in controlling the spread of vaccinia virus. The ability of a virally secreted protein to interfere with complement attack at several steps provides a mechanism for resistance ofvaccinia virus to humoral immunity. Studies by Isaacs et al. [11] suggest that this protein does indeed promote viral pathogenesis. When vaccinia virus was
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2
VCP:
-200
46 30 14
EAC43Bb in buffer and kept at O°C during the 6-min incubation period had a Z of 0.18. EAC43Bb incubated in buffer at 30°C for 6 min had a Z of 0.13, indicating that 28% of convertase activity had decayed. After cells were incubated for 6 min with purified VCP at 4 ILg/mL, Z was 0.11 (39% decay); with VCP at 8 ILg/mL, Z was 0.09 (50% decay); and with VCP at 20 lLg/mL, Z was 0.08 (56% decay). The decay rate of the alternative pathway convertase, therefore, was increased twofold when cells were incubated with VCP at 20 lLg/mL. The decay rate of the classical pathway convertase was accelerated to a similar degree at - I lLg/mL (see above). These results suggest that the decay-accelerating effect of VCP is greater on the classical pathway than on the alternative pathway. Cofactor activity for the cleavage ofC4b and C3b by factor I. No cleavage of the ex' chain of C4b occurred when EAC 14b were incubated with factor 1,0.2 lLg/mL, alone (figure 5, lane 2) or with VCP alone at 6.7 lLg/mL (lane 5) or 67 lLg/mL (lane 6). The ex' chain ofC4b was partially cleaved by factor I plus VCP at 6.7 lLg/mL (lane 3) and completely cleaved by factor I plus VCP at 67 ILg/mL (lane 4). When EAC 1423b were incubated with factor H, 170 1Lg/ mL (figure 6, lane 7), or VCP, 67 ILg/mL (lane 8), no cleavage of the C3b ex' chain was seen. On incubation with factor I,
Figure 5. Cofactor activity for cleavage ofC4b by factor I. Lane I, buffer, lane 2, factor I alone; lanes 3, 4, factor I plus VCP at 6.7 lLg/mL or 67 lLg/mL; lanes 5, 6, VCP alone at 6.7 ILg/mL or 67 lLg/mL. Positions of 0/', fl, and 'Y chains of 125I-C4b standard are shown (89 kDa, 75 kDa, and 30 kDa, respectively).
+ + + + + + + -
VCP:
NOTE. EA, sensitized sheep erythrocytes.
I:
-
1250
McKenzie et al.
Acknowledgments
We thank Ann Bjornson and Stuart Isaacs for reviewing this manuscript.
References I. Friedman HM, Cohen GH, Eisenberg Rl, Seidel CA, Cines DB. Glycoprotein C of herpes simplex virus I acts as a receptor for the C3b complement component on infected cells. Nature 1984;309:633-5. 2. Fries LF, Friedman HM, Cohen GH, Eisenberg Rl, Hammer CH, Frank MM. Glycoprotein C of herpes simplex virus I is an inhibitor of the complement cascade. 1 ImmunoI1986;137: 1636-41. 3. Kubota Y, Gaither TA, Cason 1, O'Shea 11, Lawley TJ. Characteriza-
tion of the C3 receptor induced by herpes simplex type I infection of human epidermal, endothelial, and A431 cells. 1 Immunol 1987;138:1137-42. 4. McNearney TA, Odell C. Holers VM, Spear PG, Atkinson IP. Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complementmediated neutralization of viral infectivity. J Exp Med 1987; 166:1525-35. 5. Harris SL, Frank I, Yee A, Cohen GH, Eisenberg RJ, Friedman HM. Glycoprotein C of herpes simplex virus type I prevents complementmediated cell lysis and virus neutralization. J Infect Dis 1990;162:331-7. 6. Tal-Singer R, Seidel-Dugan C. Fries L, et al. Herpes simplex virus glycoprotein C is a receptor for complement component iC3b. J Infect Dis 1991;164:750-3. 7. Mold C, Bradt BM, Nemerow GR, Cooper NR. Epstein-Barr virus regulates activation and processing of the third component of complement. 1 Exp Med 1988;168:949-69. 8. Dahlbiick B, Smith CA, Muller-Eberhard Hl. Visualization of human C4b-binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b. Proc Nat! Acad Sci USA 1983;80:3461-5. 9. Kotwal Gl, Moss B. Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins. Nature 1988;335: 176-8. 10. Kotwal Gl, Isaacs SN, McKenzie R, Frank MM, Moss B. Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 1990;250:827-30. II. Isaacs SN, Kotwal GJ, Moss B. Vaccinia virus complement-control protein prevents antibody dependent complement-enhanced neutralization of infectivity and contributes to virulence. Proc Nat! Acad Sci USA 1992;89:628-32. 12. Hammer CH, Wirtz GH, Renfer L, Gresham HD, Tack BF. Large scale isolation of functionally active components of the human complement system. 1 Bioi Chem 1981;256:3995-4006. 13. Basta M, Hammer CH. A rapid FPLC method for purification of the third component of human and guinea pig complement. 1 Immunol Methods 1991;142:39-44. 14. Rapp Hl, Borsos T, eds. Molecular basis of complement action. New York: Meredith, 1970:9-21. 15. Newman SL, Johnston RB lr. Role of binding through C3b and IgG in polymorphonuclear neutrophil function: studies with trypsin-generated C3b. 1 Immunol 1979; 123:1839-46. 16. Ellman L, Green I, Frank MM. Genetically controlled total deficiency of the fourth component of complement in the guinea pig. Science 1970;170:74-5. 17. Gaither TA, Hammer CH, Frank MM. Studies of the molecular mechanisms ofC3b inactivation and a simplified assay of I3IH and the C3b inactivator. 1 ImmunoI1979;123:1195-204. 18. Gaither T A, Frank MM. Complement. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods. Philadelphia: WB Saunders, 1984: 1245-61. 19. Kotwal Gl, Moss B. Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 1988;167:524-37. 20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
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incubated with and without antibody in the presence and absence of whole serum, virus was neutralized optimally in the presence of both antibody and complement. Complement-enhanced neutralization of antibody-sensitized virus was C4-independent and factor Be-dependent, implying a role for the alternative pathway. VCP prevented this neutralization. In normal guinea pigs, a mutant virus lacking the gene encoding VCP produced smaller skin lesions than those caused by the wild-type virus. Interestingly, similar results were obtained with C4-deficient guinea pigs. Taken together, these in vitro and in vivo experiments suggest that VCP may augment the virulence ofvaccinia virus by inhibiting a C4-independent pathway. The present report shows two possible mechanisms for this inhibition: cofactor activity to facilitate cleavage of C3b by factor I and acceleration of the decay of the alternative pathway convertase. Other viruses have evolved protective mechanisms against complement. A surface glycoprotein of herpes simplex virus, gC, binds C3b and iC3b, accelerates decay of the alternative pathway C3 convertase, and inhibits the interaction of C3b with C5 [1-6]. gC protects the virus from neutralization [4, 5]. gCl expressed on the surface of cells infected with herpes simplex virus type I protects infected cells from complement-mediated lysis by inhibiting the alternative pathway [5]. Epstein-Barr virus, another herpesvirus, has factor I cofactor activity and accelerates the decay of the alternative pathway C3 convertase [6]. Herpes simplex virus and Epstein-Barr virus possess structural proteins with complementregulatory capability. Vaccinia virus, in contrast, secretes a protein that regulates complement. The relative importance of these virulence factors has yet to be determined. Their presence suggests that further study of the function of complement in host defense against viral infection is warranted.
JID 1992; 166 (December)
Regulation of Complement Activity by Vaccinia Virus Complement-Control Protein Robin McKenzie, Girish J. Kotwal,* Bernard Moss, C. H. Hammer,* and M. M. Frank*
Laboratories ofClinical Investigation and Viral Diseases. National Institute ofAllergy and Infectious Diseases. National Institutes ofHealth. Bethesda. Maryland
The complement system is well defined as one of the principal host mechanisms defending against a wide variety of microorganisms. Most studies have centered on the role of complement in opsonization and lysis of bacteria, but complement attacks a variety of other microorganisms, including some viruses. Recently, investigators have discovered that some viruses counter this attack by producing proteins that inhibit complement activation [1-7]. Human blood contains multiple complement-regulatory proteins. These factors control activation of this crucial defense system and regulate generation of tissue-damaging protein fragments and complexes. In one large complementregulatory family, the individual proteins are made up of 4-30 short consensus repeats, each ofwhich is 60-70 amino acids long. C4-binding protein is spider-shaped, with seven 70-kDa tentacles radiating out from a central core [8]. Each tentacle contains 8 short consensus repeats. This protein binds the activated fourth component of complement, C4b, and regulates the classical complement cascade at several steps. Vaccinia virus-infected cells secrete in abundance a 35kDa protein that is structurally similar to C4-binding protein and other complement-control proteins [9]. The four short consensus repeat sequences of vaccinia complement-control protein (VCP) show marked homology to those in C4-binding protein (38%). VCP inhibits hemolysis ofsensitized sheep erythrocytes in human serum by interfering with cornple-
Received 24 January 1992; revised 22 July 1992. Reprints or correspondence: Dr. Robin McKenzie, Laboratory of Clinical Investigation, NIAID, Bldg. 10. Room l1N228. National Institutes of Health. Bethesda, MD 20892. * Present affiliations: James N. Gamble Institute of Medical Research. Cincinnati. (G.J.K.): FCRDCjNIAID. Fort Detrick, Frederick, Maryland (C.H.H.); Department of Pediatrics. Duke University Medical Center, Durham, North Carolina (M.M.F.). The Journal of Infectious Diseases 1992;166:1245-50 This article is in the public domain.
ment-mediated attack [10]. Animal studies have shown decreased virulence of a mutant virus that lacks the gene encoding VCP [II]. We investigated the mechanisms by which VCP inhibits complement activity, thereby increasing the pathogenicity of vaccinia virus.
Materials and Methods Buffirs and reagents. The following buffers were used: isotonic veronal buffered saline with 0.1%gelatin (GVBS); GVBS with 0.15 mM calcium chloride and 1.0 mM magnesium chloride (GVBS++); GVBS++ diluted 60:40 with 5% dextrose containing 0.1% gelatin, 0.15 mM calcium chloride, and 1.0 mM magnesium chloride (DGVBS++); GVBS with 20 mM EDT A (EDTA-GVBS); GVBS with 10 mM EGTA and 2 mM magnesium chloride (Mg/EGTA); Mg/EGTA diluted 60:40 with 5% dextrose containing 0.1% gelatin, 10 mM EGT A, and 2 mM magnesium chloride (60% mg/EGTA buffer); PBS; and Alsever's solution (0.11 M dextrose, 27 mM sodium citrate, 72 mM sodium chloride). Sources ofcomplement components. Human C4 and C3 were purified by the method of Hammer et al. [12] modified to include passage over a fast protein liquid chromatography anion exchange column (Fast Q; Pharmacia, Piscataway, NJ) [13]. Final concentrations of C4 and C3 were 70 and 500 JLg/mL, with activities of 40,000 and 50,000 units/ml., respectively. One unit was defined as the reciprocal of the dilution required to produce 63% lysis of antibody-coated sheep red blood cells at 1.5 X 108 cells/rnl. [14]. Purity assessed by Coomassie blue staining of7.5% SDS-polyacrylamide gels was >95%. To cleave C4 and produce C4b, fractions from the Fast Q column shown to contain C I protease activity were incubated at 37°C overnight. Paranitrophenyl guanidinobenzoate (Sigma, St. Louis) and aprotinin (Sigma) were then added before storage at -85°C. C3 was converted to C3b by incubation with varying concentrations of trypsin for 3 min at 37°C [15]. Proteolysis from these two methods was then assessed by SDS-PAGE. Guinea pig C I and human C2 were purchased from Diamedix (Miami). Serum from guinea pigs genetically deficient in C4
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A major protein secreted by vaccinia virus-infected cells has structural similarity to the superfamily of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.
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McKenzie et al.
Richmond, CA) using a NaCl gradient followed by passage through Sephadex G-100 (Pharmacia) with GVBS++ as the buffer [10]. The protein concentration ofthe final eluant was 67 ~g/mL as determined by bicinchoninic acid assay (Pierce, Rockford, IL). Purity was assessed by SDS-PAGE. Radiolabeling. Purified C3, purified C4, and concentrated medium (300X) from vaccinia virus-infected cells were radiolabeled with 1251 using lodobeads (Pierce) according to the manufacturer's instructions. The labeled proteins were separated from free iodine using a PD-I 0 column (Pharmacia) eluted with PBS. Specific activities of C3 and C4 were 2.2 X 106 and 1.3 X 106 cpm/ug, respectively. Five percent bovine serum albumin was added to the purified proteins before storage at -70°C. SDS-PAGE. SDS-PAGE was done on 7.5% and 12% gels under reducing conditions [20]. Autoradiograms were analyzed by densitometry using an Ultroscan XL laser densitometer (LKB, Piscataway, NJ). Binding to C4b- and C3b-Sepharose. C4b, C3b, and human serum albumin were coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. Protein (5-10 mg) was coupled to each milliliter of beads. 1251-labeled medium from vaccinia virus-infected cells was applied to columns of C4b- or C3b-Sepharose. After thorough washing with DGVBS++, columns were eluted with DGVBS++ containing I M NaCI. Eluants were desalted on PD10 columns (Pharmacia) using PBS as the buffer. Lysis ofEAC14b in C4D serum. Since C4D serum provides all complement components except C4, EACI4b will lyse in C4D serum. To determine whether VCP would block this lysis, EAC 14b ( 1.5 X 108 /mL, made with about one active hemolytic C4b site per cell) were incubated for 30 min at 37°C with either medium from cells infected with vaccinia virus, medium from cells infected with the 6/2 mutant, or purified VCP. Then C4D serum diluted 1:75 in DGVBS++ was added. After 60 min, lysis of erythrocytes was determined by reading the optical density at 412 nm. Effect on classical C3 convertaseformation. C4-binding protein inhibits the formation of the classical C3 convertase. Experiments were, therefore, done to examine the effect of media from vaccinia virus-infected cells on the formation of C 142a, the classical C3 convertase. Increasing concentrations ofC2 in a volume of I00 ~L were added to tubes containing I00 ~L of either DGVBS++ buffer, a IX or Y3X concentration of medium from vaccinia virus-infected cells, or a IX concentration of medium from cells infected with the vSIGK3 mutant virus. EAC14b (100 ~L at 1.5 X 108/mL, made with limited C4) were added. Tubes were incubated for 4 min at 30°C. ComplementEDT A (I mL) was then added, and incubation continued for an additional 60 min at 37°C. After centrifugation, lysis was quantitated by measuring the optical density of the supernatants at 412 nm. Results were expressed as Z, the number of hemolytically active sites per erythrocyte. Effect on classical C3 convertase decay. C4-binding protein accelerates the decay of EACI42a. Experiments were done to determine if VCP affects this rate of decay. EAC 14b (I 00 ~L at 1.5 X 108/mL, made with about one hemolytically active C4b site per cell) were incubated with I00 ~L ofC2, 20 units/rnl., for 4 min at 30°C. Cells were washed with ice-cold DGVBS++ and
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(C4D serum) obtained from our own colony of guinea pigs [16] contained no C4 by hemolytic assay. Complement-EDTA, used as a source of C3-C9, was prepared by incubation of normal guinea pig serum in EDTA-GVBS at a I:25 dilution for 15 min at 3rc. (EDTA chelates calcium and magnesium needed for activation of Cl , C4, and C2 but does not interfere with activation ofC3-C9.) Factors I, B, and D were purchased from Quidel (San Diego). Factor H was purified as reported [17]. Cellular intermediates. Complement can be activated by the classical and alternative pathways. Each of these pathways produces a C3 convertase that activates C3 and then C5-C9. Activation of the complement components through C9 on the surface of a sheep erythrocyte causes lysis of the erythrocyte. C142a, the classical C3 convertase, and C43Bb, the alternative pathway C3 convertase, were prepared as follows. Sensitized sheep erythrocytes (EA) were used as targets to activate and bind complement components. EA were sequentially incubated with purified C I followed by C4 to produce EACI4b and further incubated with C2 to produce EACI42a [18]. EAC 14b with limited C4 sites were prepared by incubating EAC I (1.5 X 108 /mL) in DGVBS++ containing I unit/rnl. purified C4. EAC43b were made by incubating EACI (5 X 108/mL) first with C4, 33 units/ml., for 45 min at 37°C and then with C2, 300 units/rnl., and C3, 450 units/ml., for 30 min at 37°C. Cells were then incubated in 10 mM EDT A for two 20-min incubations at 37°C. The resulting EAC43b did not lyse when 0.1 mL of cells was incubated for I h at 37°C in I mL of complementEDTA, showing the absence ofC3 convertase activity. EAC43Bb were formed by incubating EAC43b (2.4 ml., 1.5 X 108 cells/ml.) with varying amounts of factor B and factor D for 30 min at 30°C. To calculate the number of hemolytically active sites per cell, 0.1 mL ofEAC43Bb was added to I mL of complement-EDTA and the mixture incubated for I h at 37°C. After centrifugation, the optical density of the supernatants was read at 412 nm and the percentage oflysis (y) determined. The average number of hemolytically active sites per cell (2) was then determined according to the formula: Z = -In(1 - y) [14]. Incubation of EAC43b with 60 ng of factor Band 400 ng of factor D produced EAC43Bb with about one active site per cell. Viral culture and purification of vCP. RK 13 cells (CCL 37; American Type Culture Collection, [ATCC], Rockville, MD) in serum-free OPTI-MEM I medium (GlBCO, Grand Island, NY) were infected with 5 pfu/cell of either wild-type vaccinia virus (strain WR, VR1354; ATCC) or one of two mutants: 6/2, a spontaneous mutant lacking a cluster ofgenes including the one that encodes VCP [19], or vSIGK3, a recombinant mutant with a 70-bp segment within the VCP gene replaced by the selectable marker gene, xanthine-guanine phosphoribosyltransferase, regulated by a vaccinia promoter [10]. After a l-h incubation, the monolayer was washed with serum-free OPTI-MEM and reincubated in this medium for 24 h. Tissue culture medium from infected cells was then centrifuged at 2000 g for 10 min, concentrated 25x-300X using a Centricon 30 (Amicon, Danvers, MA), and diluted in DGVBS++ before use. Concentrations are expressed relative to unconcentrated, undiluted tissue culture medium. VCP was isolated from concentrated medium by two chromatographic procedures: elution from DEAE-Biogel (Bio-Rad,
JID 1992;166 (December)
JID 1992; 166(December)
Vaccinia Complement-Control Protein
1247
Results Figure 1. Binding of VCP to C4b- and C3b-Sepharose. SDSPAGE and autoradiography of (2SI-labeled tissue culture me- 20093dium (lanes I, 5), column washes 69(lanes 2,3,6,7), and desalted col- 46umn eluants (lanes 4,8) show 35kDa bands in high-salt eluents 30but not washes. 14-
100
80
"U
60
e
~
0 Gl
.,.
40
20
0 0
20
40
60
80
100
Time (min)
Figure 2. Binding of purified 12sI-labeled VCP to C4b-Sepharose at timed intervals.
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resuspended in this buffer at 1.5 X 108/m L. The resulting EACI42a were allowed to decay at 30°C during incubation with buffer, with medium from vaccinia virus- or 6/2 mutant virus-infected cells, or with purified VCP at a final concentration of 1.3 or 0.67 ILg/mL. At timed intervals, 0.2 mL of the cell suspension was mixed with I mL ofcomplement-EDTA and the mixture incubated at 37°C for 60 min. After centrifugation, the optical density of the supernatant was measured at 412 nm. Effict on alternative pathway convertase decay. EAC43Bb, 0.1 mL, made as above were incubated for 6 min at 30°C with 0.1 mL ofDGVBS++ or VCP at concentrations indicated. Complement-EDTA (I mL) was added and the mixture incubated an additional 60 min at 3rc. The optical density at 412 nm was then measured. Regeneration of classical C3 convertase activity after decay. To determine whether the classical C3 convertase was reversibly or irreversibly altered during decay with VCP, C2 was added back to the convertase after decay. The C42a convertase activity ofEACI42a (1.5 X 108/mL) prepared with limited C4 was allowed to decay in the presence of DGVBS++ buffer or medium from vaccinia-infected cells (3X final concentration). As a control, EACI4b were incubated with buffer. After 30 min, cells were washed twice in cold DGVBS++,and remaining C42a sites were quantitated as above by incubating 200 ILL of each cell suspension with I mL of complement-EDTA and measuring lysis of cells. To regenerate decayed C42a sites, another aliquot of the cell suspension after decay was mixed with C2 at 50 units/ mL. After incubation at 30°C for 4 min, cells were washed and added to complement-EDTA to quantitate hemolytically active C42a sites. Cofactor activity for the cleavage of C4b and C3b by factor I. In the presence of a cofactor such as factor H or C4-binding protein, factor I cleaves C3b and C4b. Experiments were done to see if VCP has similar cofactor activity. EACI4b and EACI423b prepared with 12sI-labeled C4 and 12sl-labeled C3, respectively, were incubated for 60 min at 37°C with factor I, VCP, or factor H and with combinations of these factors at the concentrations indicated. Cells were then washed in DGVBS++. EACI423b were incubated for I h at 37°C in 0.025 M methylamine buffer containing 0.05 M sodium bicarbonate, 1% SDS, and 25 ILM paranitrophenyl guanidinobenzoate (Sigma) to free ester-bound radiolabe1ed C3 from the erythrocyte. After neutralization with 0.1 N HCI, these samples and the EAC 14b were boiled in SDS buffer containing 2-mercaptoethanol and analyzed by SDS-PAGE.
Binding to C4b- and C3b-Sepharose. Medium from cells infected with wild-type vaccinia virus was concentrated, labeled with 1251, and applied to columns packed with C4b- or C3b-Sepharose. Columns were washed with DGYBS++ and then eluted with DGVBS++ containing 1 M saline. SDSPAGE of the high-salt eluants showed a predominant band with the expected molecular mass ofYCP, 35 kDa (figure 1). In a timed experiment, most binding to C4b-Sepharose occurred within 5 min, with no further increase in binding after 30 min (figure 2). To determine whether the material eluted from the C4b-Sepharose column would bind to C3b, the eluant was desalted and added to duplicate tubes containing either C4b-Sepharose beads, C3b-Sepharose beads, or human serum albumin-Sepharose beads. Tubes were rotated at 4°C for 30 min. Seventy-five percent of the 1251-labeled material was rebound by the C4b-Sepharose, 60% was bound by the C3b-Sepharose, and 13% by the control, human serum albumin-Sepharose. A decrease in rebinding to C4b-Sepharose may have been due to less efficient contact between YCP and C4b in rotating tubes or to some denaturation of the protein. The ability of most of the radioactive material eluted from the C4b column to rebind to C3bSepharose and the similarity in the bands on SDS-PAGE (figure 1) indicate that the same 35-kDa protein binds to both C3b and C4b. Inhibition of the lysis ofEAC14b in C4D serum. Lysis of EAC 14b in C4D serum is inhibited in a dose-response fashion by medium from cells infected with vaccinia virus but not by medium from cells infected with the 6/2 mutant [10]. In the current experiments, 50% inhibition occurred when vaccinia virus medium was present at a final dilution of I in
McKenzie et al.
1248
0.2-r--------------,
z
0.1
40
C2
60
80
'00
Buffer
--0--
vSIGK3, 1X
---0-
WR. 113X
____
WR,lX
120
(units/mil
Figure 3. InhibitionofC42a convertase formation on sensitized sheep erythrocytes. WR, medium made from cells infected with wild-type vaccinia virus; vSIGK3, medium fromcellsinfectedwith vSIGK3 mutant virus. Z. mean number of hemolytically active sites per erythrocyte.
4. Lysis was also inhibited by purified VCP. About 50%inhibition occurred when VCP was present at a final concentration of 4 ~g/mL. No inhibition was produced by medium from cells infected with the 6/2 mutant. To determine whether preincubation ofEACl4b with the vaccinia protein was necessary to produce inhibition, cells were incubated with medium from vaccinia virus-infected cells for 0-30 min at 37°C or O°C before the addition of C4D serum. No change in the percentage of inhibition occurred under these conditions. Inhibition ofclassical C3 convertaseformation. EACl4b with limited amounts of C4b were incubated with increasing amounts of C2 together with buffer, a 1X or Y3X concentration of medium from vaccinia virus-infected cells, or a 1X concentration of medium from vSIGK3 virus-infected cells. Classical pathway C3 convertase activity was then determined by further incubation with complementEDT A. Formation of the convertase was inhibited by medium from wild-type vaccinia virus but not by medium from the mutant virus at all concentrations of C2 tested (figure 3). An additional experiment was done to see whether interaction of VCP with EAC 14b before addition of C2 was required for the inhibitory effect. In one set of duplicate tubes, EAC 14b were incubated first with 1X vaccinia virus medium for 4 min, followed by the addition of excess C2 (10 units/ mL) for an additional 4 min of incubation. In a second set of tubes, EAC 14b were incubated first with C2 for 4 min before the addition of 1X medium for another 4 min. In a third set of tubes, EAC 14b were incubated with C2 and 1X medium together for 8 min. Under these three conditions, inhibition was 56%, 56%, and 54%, respectively, showing no difference. Acceleration of classical C3 convertase decay. EACl4b
made with about one hemolytically active site per cell were incubated with C2, 20 units/rnl., for 4 min at 30°C. The EACl42a were washed, then incubated with buffer or with medium from vaccinia virus- or 6/2 virus-infected cells. At timed intervals, 0.2 mL of the cell suspension was mixed with 1 mL of complement-EDTA, the mixture incubated at 37°C for 60 min, and lysis measured. When EAC 142a were incubated in buffer or medium from 6/2-infected cells diluted 1 in 2, the half-life of the C42a enzyme was 7 min. When medium from vaccinia virusinfected cells was present at a dilution of 1 in 6.7 or 1 in 2.5, the half-life decreased to 2 min and 1 min, respectively. Medium from cells infected with the 6/2 mutant produced no change in the decay rate. Purified VCP also accelerated the decay of the C42a convertase. When the protein was present at a final concentration of 0.67 ~g/mL or 1.3 ~g/mL, the half-life decreased to 4 min and 3 min, respectively (figure 4). Experiments were done to determine whether VCP destroyed the C4b site. After decay in the presence of either buffer or medium from vaccinia virus-infected cells, cells were washed in cold buffer. Convertase activity could then be completely restored by the addition offresh C2 (table 1). VCP, therefore, accelerates classical convertase decay by a reversible, nonproteolytic mechanism. Acceleration of alternative pathway convertase decay. Aliquots of EAC43Bb with a limited number of hemolytically active sites per cell were incubated for 6 min at 30°C with varying concentrations of purified VCP or DGVBS++. Complement-EDTA was then added and samples incubated at 37°C for 60 min. Hemolysis was quantitated by measuring the optical density at 412 nm. A control sample containing
10.,....
-,
z .1
0
BUFFER
&
VCP 0.67 UGIML
I
I
01
L~
o
10
20
vCP 1.3 UGIML
30
TIME (MIN)
Figure 4. Acceleration of C42a convertase decay on sensitized sheep erythrocytes by VCP.
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20
____
JID 1992; 166 (December)
Vaccinia Complement-Control Protein
110 1992; 166(December)
1249 2345678
Table 1. Reconstitution ofC42 convertaseafter decay in the presence of medium from vaccinia virus-infected cells. I: H:
C42 convertase activity (2)
Cells EACl42 EACl42 EACl4
Reagen t present during decay
After decay
After reconstitution withC2
Buffer 3x vaccinia medium Buffer
0.11 0 0
1.48 1.49 1.49
a{3-
a'{J-
y-
-
93 69
- +
3 4 5 6
+ + ....III
~
--~
Ql C
~
....III Ql
c
Figure 6. Cofactor activity for cleavage of C3b by factor I. Lane I, buffer;lane 2, factor I alone; lanes 3-5, factor I plus VCPat"0.67 ILg/mL, 6.7 lLg/mL, or 67 lLg/mL; lane 6, factor I plus factor H; lane 7, factor H alone; lane 8, VCP alone at 67 ILg/mL. Positions of 0/ and fl chains of nonactivated C3 standard are shown (120 kDa and 75 kDa, respectively). Molecular mass ofC3b 0/ chain is 110 kDa. Degradation of C3b 0/' chain produces 0/'1 (68 kDa) and 01'2 (43 kDa); O/'! chain is further degraded to 0/'3 (27 kDa) and 01'4 (41 kDa).
0.03ILg/mL (lane 2), or with factor I plus VCP at 0.67 ILg/mL (lane 3), 7% of the ex' chain was cleaved. Factor I, together with VCP at 6.7 ILg/mL (lane 4) or 67 ILg/mL (lane 5), cleaved 21 % and 57% of the 0/' chain, respectively. Factors I and H together (lane 6) cleaved 71 % of the 0/' band.
Discussion VCP, a major secretory protein of vaccinia virus, is structurally homologous to C4-bindtng protein [9]. Like C4-binding protein, VCP binds to C4b and regulates the classical complement pathway. This regulation controls the formation and decay ofthe classical C3 convertase and the degradation of C4b by factor I. These effects occur with concentrations ofVCP present in un concentrated, unpurified medium from vaccinia virus-infected cells. Similar to other complement regulatory proteins, VCP affects the alternative pathway. It binds to C3b and acts as a cofactor with factor I in the degradation of C3b. Although VCP accelerates the decay of the alternative pathway convertase, under the conditions examined higher concentrations were required than were needed to affect the classical pathway convertase. It is generally believed that cellular immunity is more prominent than humoral immunity in controlling the spread of vaccinia virus. The ability of a virally secreted protein to interfere with complement attack at several steps provides a mechanism for resistance ofvaccinia virus to humoral immunity. Studies by Isaacs et al. [11] suggest that this protein does indeed promote viral pathogenesis. When vaccinia virus was
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2
VCP:
-200
46 30 14
EAC43Bb in buffer and kept at O°C during the 6-min incubation period had a Z of 0.18. EAC43Bb incubated in buffer at 30°C for 6 min had a Z of 0.13, indicating that 28% of convertase activity had decayed. After cells were incubated for 6 min with purified VCP at 4 ILg/mL, Z was 0.11 (39% decay); with VCP at 8 ILg/mL, Z was 0.09 (50% decay); and with VCP at 20 lLg/mL, Z was 0.08 (56% decay). The decay rate of the alternative pathway convertase, therefore, was increased twofold when cells were incubated with VCP at 20 lLg/mL. The decay rate of the classical pathway convertase was accelerated to a similar degree at - I lLg/mL (see above). These results suggest that the decay-accelerating effect of VCP is greater on the classical pathway than on the alternative pathway. Cofactor activity for the cleavage ofC4b and C3b by factor I. No cleavage of the ex' chain of C4b occurred when EAC 14b were incubated with factor 1,0.2 lLg/mL, alone (figure 5, lane 2) or with VCP alone at 6.7 lLg/mL (lane 5) or 67 lLg/mL (lane 6). The ex' chain ofC4b was partially cleaved by factor I plus VCP at 6.7 lLg/mL (lane 3) and completely cleaved by factor I plus VCP at 67 ILg/mL (lane 4). When EAC 1423b were incubated with factor H, 170 1Lg/ mL (figure 6, lane 7), or VCP, 67 ILg/mL (lane 8), no cleavage of the C3b ex' chain was seen. On incubation with factor I,
Figure 5. Cofactor activity for cleavage ofC4b by factor I. Lane I, buffer, lane 2, factor I alone; lanes 3, 4, factor I plus VCP at 6.7 lLg/mL or 67 lLg/mL; lanes 5, 6, VCP alone at 6.7 ILg/mL or 67 lLg/mL. Positions of 0/', fl, and 'Y chains of 125I-C4b standard are shown (89 kDa, 75 kDa, and 30 kDa, respectively).
+ + + + + + + -
VCP:
NOTE. EA, sensitized sheep erythrocytes.
I:
-
1250
McKenzie et al.
Acknowledgments
We thank Ann Bjornson and Stuart Isaacs for reviewing this manuscript.
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incubated with and without antibody in the presence and absence of whole serum, virus was neutralized optimally in the presence of both antibody and complement. Complement-enhanced neutralization of antibody-sensitized virus was C4-independent and factor Be-dependent, implying a role for the alternative pathway. VCP prevented this neutralization. In normal guinea pigs, a mutant virus lacking the gene encoding VCP produced smaller skin lesions than those caused by the wild-type virus. Interestingly, similar results were obtained with C4-deficient guinea pigs. Taken together, these in vitro and in vivo experiments suggest that VCP may augment the virulence ofvaccinia virus by inhibiting a C4-independent pathway. The present report shows two possible mechanisms for this inhibition: cofactor activity to facilitate cleavage of C3b by factor I and acceleration of the decay of the alternative pathway convertase. Other viruses have evolved protective mechanisms against complement. A surface glycoprotein of herpes simplex virus, gC, binds C3b and iC3b, accelerates decay of the alternative pathway C3 convertase, and inhibits the interaction of C3b with C5 [1-6]. gC protects the virus from neutralization [4, 5]. gCl expressed on the surface of cells infected with herpes simplex virus type I protects infected cells from complement-mediated lysis by inhibiting the alternative pathway [5]. Epstein-Barr virus, another herpesvirus, has factor I cofactor activity and accelerates the decay of the alternative pathway C3 convertase [6]. Herpes simplex virus and Epstein-Barr virus possess structural proteins with complementregulatory capability. Vaccinia virus, in contrast, secretes a protein that regulates complement. The relative importance of these virulence factors has yet to be determined. Their presence suggests that further study of the function of complement in host defense against viral infection is warranted.
JID 1992; 166 (December)
