Clin. exp. Immunol (1987) 67, 531-536

Antibody-independent activation of the classical pathwayof complement by Epstein-Barr virus

H. MARTIN, I. McCONNELL*, B. GORICKt & N. C. HUGHES-JONESt MedicalResearch Council, Mechanisms in Tumour Immunity Unit, MRC Research Centre, Cambridge;* Royal School of Veterinary Studies, Edinburgh; t Agricultural Research Council Institute of

Animal Physiology, Babraham, Cambridge

(Acceptedfor publication 29 September 1986)

SUMMARY

A purified preparation of Epstein-Barr virus (EBV) has been shown to activate theclassical complement pathway by direct interaction with the first component ofcomplement, Cl, without the intervention of antibody. No evidence was found foractivation of the alternative pathway. Following classical pathway activation the specificaffinity ofEBV for B cells can be presumed to be lost since the virus will become opsonizedfor clearance by phagocytic cells bearing complement receptors, CR1 and CR3. Thisactivation is further evidence that complement plays a role in defence mechanismsindependently of antibody activity.

Keywords Epstein-Barr virus C1

INTRODUCTION

It is known that the classical pathway of complement can be activated by a variety of non-immunological mechanisms. Acidic mucopolysaccharides, polyanions, heparin and glutaralde-hyde-treated red cells can all directly interact with Clq to initiate classical pathway activation(Loos, 1982).

In addition it is also known that certain animal retroviruses can directly activate the classicalpathway through the interaction of a viral polypeptide with Clq (Cooper et al., 1976). We havepreviously shown that Epstein-Barr virus (EBV)-transformed lymphocytes activate humancomplement by the alternative pathway without involving antibody (McConnell et al., 1978). Wenow report that purified EBV can itself activate the classical pathway by direct interaction with Clwithout involving antibody.

MATERIAL AND METHODS

Epstein-Barr virus was purified from the supernatant of the marmoset B95 8 cell line which hadbeen cultured in the presence of 20 ng/ml of the tumour promoter, TPA. Epstein-Barr (250-500 pgof protein) was purified from 5 1 of supernatant by ultracentrifugation and density gradientfractionation on Dextran T-10 (Pharmacia, Sweden) (Nemerow & Cooper, 1981). In thispurification the virus-containing fractions were detected by spot hybridization on nitrocelluloseusing a radiolabelled EBV DNA probe (kindly supplied by Dr B. Barrel, MRC Laboratory ofMolecular Biology, Cambridge).

Correspondence: Dr H. Martin, Institut fur Medizinische Mikrobiologie, Hochhaus am Augustusplatz,6500 Mainz, FRG.

53I

Electron-miroscopic examination of the virus revealed typical EBV particles and the biologicalactivity of the virus was checked by its ability to transform human B lymphocytes intolymphoblastoid cell lines. Biochemical characterization by silver stained SDS-PAGE revealed thetypical pattern of the high molecular weight polypeptides of EBV (Morrisey, 1981). Thepreparation contained trace amounts of albumin but no other protein bands associated with fetalcalf serum or with B cells. The EBV preparations were free of mycoplasma as judged by electronmicroscopy and by failure to culture the organisms.

Extent of C3 conversion in serum. Epstein-Barr virus (10 ug) was incubated with 20 yul ofhypogammaglobulinaemic human serum for 30 min. A control consisted of hypogammaglobuli-naemic serum incubated without EBV. Classical pathway was examined in the presence of Ca (ImM) and Mg (1 mM) and the alternative pathway in the presence ofMg (7 mM) and EGTA (10 mM).Rocket immunoelectrophoresis using an anti-C3 serum was carried out by the method of Laurell(1966). The hypogammaglobulinaemic human serum contained < 500 pg/ml IgG, < 200 ug/ml IgAand < 100 pg/ml IgM and contained no antibody activity against EBV-associated antigens (VCA,EA or EBNA).

Cl activation. Human Cl was purified according to the method of Gigli, Porter & Sim (1976)and iodinated by the lactoperoxidase method of Heusser et al. (1973). Cl activation was measuredas described by Hughes-Jones & Gorick (1982). Briefly, the test sample (e.g. 20 pg of EBV) wasincubated with 0 6 pg of purified 251I-labelled Cl and incubated at 370C for times ranging from 0 to40 min. The reaction was stopped by adding SDS sample buffer containing 2% mercaptoethanoland boiling. Samples were run reduced on a 10% SDS polyacrylamide gel which was then drieddown and autoradiographed. The extent ofCl activation (i.e. loss of intact 83 kD C I r and C I s andconcomitant gain of Cl r and Cl s heavy chain and Cl r light chain fragments) was quantified bydensitometry. The light chain of CIs molecule does not take up 1251 during the labelling procedure.Similarly the b-chain of Clq is not labelled and so only the a and c chains are detectable byautoradiography.

Proteolytic cleavage ofpurified '251I-labelled C3. Purified human C3 was kindly supplied by Dr R.Harrison (MITI unit, MRC Centre, Cambridge). Purity was demonstrated by SDS-PAGE andCoomassie staining which revealed the presence only of the C3 a (126 kD) and / (72 kD) chains.Four hundred ng C3, labelled with '251 according to Fraker & Speck (1978), was incubated forvarious time intervals swith either 10 pg EBV or 10 ng trypsin. The mixtures were then analysed byreduced SDS-PAGE and C3 a chain cleavage was visualized by autoradiography.

RESULTS

Activation of classical pathway by EBV. Figure I shows that incubation of purified EBV withhypogammaglobulinaemic human serum under conditions supporting either classical or alternativepathway activation resulted in marked activation of the classical but not the alternative pathway.Activation was shown by measuring the extent ofC3 conversion with 10 pg ofEBV and it was foundthat there was approximately a 50% conversion of C3 to iC3b in 30 min. No activation wasobserved under conditions which only supported alternative pathway activation.

This is quite distinct from the situation with EBV-transformed cells which produce markedactivation under conditions in which only the alternative pathway could be activated (McConnell etal., 1978).

Activation of 125S-labelled Cl. In order to exclude further any role for antibody in classicalpathway activation, purified EBV was incubated with purified '25I-labelled Cl and the extent ofcleavage ofCl r and C I s determined. As shown in Figs 2 and 3 purified EBV (EBV (a)) was able toproduce activation of 75% of the Cl after 40 min incubation. A partially purified EBV preparation(EBV (b)) also produced Cl activation under the same circumstances (Fig. 3). A small amount ofauto-activation of the Cl was observed in controls containing Cl alone (Figs 2 and 3) and in thepresence of fetal calfserum (Fig. 3). Some ofthe Cl is already activated at the start ofthe experiment(estimated by densitometry to be about 18%, Fig. 3). The already cleaved C I r and C I s is detected asa band at 56 kD. The CIr light chain produced by the same cleavage is not detectable until about

532 H. Martin et al.

Classical complement pathway and LBV 533Classical Alternativepathway pathwayC3 C3b C3b CU3

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Fig. 1. Analysis of C3 conversion to iC3b by ERV through the classical pathway by rocket immunoelectrophore-sis using an anti C3 serum. The addition of zymosan to the serum showed that the alternative pathway could beactivated. Control is serum incubated alone without EBV.

40% activation is achieved (see EBV (a) 10 mmn incubation Figs 2 & 3) because the heavy chainportions ofCl1r and Ci1s take up the majority of the radiolabel duning the iodination procedure. TheCls light chain portion and the b subunit of Clq are not labelled and so do not appear on theautoradiograph.

Absence ofprotease in the EBVpreparation. It is possible that C3 conversion could have been dueto the presence of a protease contaminant in the EBV preparation. The splitting of purified C3 is ahighly sensitive test system, since it can be cleaved by as little as 10 ng of trypsin. There was noevidence of protease contamination, as the addition of 10 pg of EBV did not cause any breakdownof purified C3 (Fig. 4).

DISCUSSION

These experiments show that the antibody independent activation of the classical pathway seen inwhole serum following incubation with EBV (Fig. 1) is due to direct interaction of the virus with Cl

a.~~~~~~ I I . ww%oa

H. Martin et al.EBV(a)

Clr,Cls

H ClsH CIr

L CIr

______--_-----

Self act0 10 20 40 0 10 20 40

Time (min) Time (min)Fig. 2. Autoradiograph of SDS-PAGE of I251-labelled Cl after incubation with EBV (EBV (a), right hand side)or on its own (self act., left hand side). C activation is seen by the conversion of the 83,000 mol. wt CIr and CIs

molecules to their heavy (56,000 mol. wt) and light chain (27,000 mol. wt) derivatives. The light chain runs at the37,000 mol. wt position in polyacrylamide gels although its true molecular weight is 27,000. A small percentageof the Cl is already activated as shown by the presence of the 56 K band. The 37 K band does not appear untilaround 40% activation because the Clr light chain portion is only weakly labelled.

80

0

400

20

0

EBV (a)

EBV (b)

/. FCS

Self-act

20 40Time (min)

Fig. 3. Densitometric quantification of the degree of Cl activation shown in Fig. 2. In addition to the highlypurified EBV (EBV (a)) and the negative control containing no additional material (self act), a partially purifiedEBV preparation (EBV (b)) and fetal calfserum (FCS) were tested for C activation by the same method. Thereis a background activation during purification and labelling of the molecule.

(Figs 2 & 3). The most probable mechanism is that Clq combines directly with a protein on the viralenvelope and this reaction then results in the activation ofClr-CI s tetramer by a process which hasyet to be defined. This mechanism has been described for retro-viruses which activate the classicalpathway in human serum, where it is known that Clq directly binds to the p15 E polypeptide ofmurine retro-viruses (Bartholomew & Esser, 1978). It is unlikely that activation resulted from a

534

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a

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I

Classical complement pathway and LI V 535C3 C3 C3

1 alone I +EBV I + trypsin I Mr

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10 30 90 10 30 90 10 30 90Time {min)

Fig. 4. Evidence for absence of protease in the EBV preparation. Ten micrograms of EBV was incubated with400 pg of purified '251-labelled C3 for 10, 30 and 90 mmn at 37 C before SDS-polyacrylamide electrophoresis,followed by autoradiography. Controls consisted of(a) C3 alone and (b) C3 with 10 ng of Trypsin. The 10 ng oftrypsin resulted in almost complete conversion of the 126 kD alpha chain of C3 into the 116 kD fragments within30 mi, whereas 10 jg of EBV was without enzymic activity.

protease present in the EBV preparation, since none could be detected using the splitting of C3 as ahighly sensitive test system (Fig. 4).

It has been convincingly demonstrated that the EBV receptor on B cells is the CR2 molecule, thereceptor for C3d,g. Jondal et al. (1976) showed that EBV receptors and C3 receptors werecoincidentally expressed on B cells.

Identity between CR2 and EBV receptors was demonstrated by Jdnsson, Wells & Klein (1982).Nemerov, Wolfert & Cooper (1984) found evidence to suggest that the 145 kD CR2 proteincontains separate domains, one to which EBV binds and the other to which C3d,g binds.

Through classical pathway activation the EBV particle may become coated with C3 which willbe cleaved subsequently to C3d,g. However, it is most unlikely that such complement activationleads to enhanced viral infectivity by facilitating binding to the B cell CR2. The EBV particle, beforeinteraction with complement, already possesses a specific affinity for B cells by virtue of therestricted distribution of CR2 to this cell type. After interaction with complement, this specificitycan be presumed to be lost since both C3b and C3bi will appear on the virus surface long beforeexposure of C3ds,g. Thus, antibody independent classical pathway activation most probablyrepresents a natural host defence mechanism which facilitates elimination of virus.

It has been shown with enveloped viruses such as influenza virus or simian virus 5 that theirabilities to activate the alternative pathway are dependent on the cell lines from which these virusesare derived (McSharpy, Pickering & Caliguiri, 1981). Variation in the degree of sialation ofthe EBVmembranes released from B95 8 sublines may explain the finding by Mayes, Nemerov & Cooper(1983) that the purified EBV preparation activated the alternative complement pathway whereasthe biologically active purified virus used in this study gave no evidence for alternative pathwayactivation (Fig. 1). Thus, it remains unclear to what extent alternative pathway activation by EBVrepresents a natural phenomenon. Figures 1-3 are the first experiments which test for C I activationby direct interaction with EBV. It is known that classical pathway components do not neutralize theEBV (Nemerow, Jensen & Cooper, 1982). The results described here provide a more direct andsensitive assessment of interaction of EBV with the classical pathway than failure to neutralizeinfectivity.

Nemerow & Cooper (1981) and Nemerow et al. (1982) report that specific XEBV antibody orsmall quantities of crossreacting anti-Herpes simplex type 1 virus antibodies, in the presence of C 1,

_w I I I I 7 7 blotT

536 H. Martin et al.C4, C2, and C3, were able to neutralize EBY infectivity. In addition, they found that noneutralization occurs under conditions which support either antibody-independent classicalpathway or alternative pathway activation. Mayes et al. (1983) report that purified EBV activatesthe alternative pathway of complement. Thus, antibody independent complement activation byEBV does not imply that neutralization should occur. Specific antibodies may be required to directthe binding of complement proteins to sensitive viral structures. Therefore, the failure of theclassical pathway to neutralize EBV infectivity in absence of antibody (Nemerow et al., 1982) doesnot contradict our demonstration of antibody independent classical pathway activation by EBV.The sensitive and direct assessment of EBV interaction with the classical pathway presented in Figs1 and 2, in conjunction with the observation by Nemerow et al. (1982) that classical pathwaycomponents alone are insufficient to neutralize EBV, suggests that the outcome of antibodyindependent classical pathway activation is opsonization of the virus.

It is becoming increasingly clear that complement plays a role in defence mechanisms againstpathogens independently of antibody activity. The antibody-independent destruction oforganismshas been established not only for mouse leukaemia virus, but also for Salmonella Minnesota (Clas &Loos, 1981), Klebsiella species, Escherichia coli (Loos et al., 1978) and probably Mycoplasmapneumoniae (Bredt et al., 1977). We are currently investigating the role of antibody independentpathway activation in the opsonization of EBV.

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