Cell surface activation of the alternative complement pathway by the fusion protein of measles virus

Patricia Devaux, Dale Christiansen, Sébastien Plumet and Denis Gerlier

Immunité & Infections Virales, CNRS-UCBL UMR 5537, IFR 62 Laennec, Rue Paradin, 69372 Lyon Cedex 08, France

Correspondence
Denis Gerlier
gerlier{at}laennec.univ-lyon1.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Measles virus (MV)-infected cells are activators of the alternative human complement pathway, resulting in high deposition of C3b on the cell surface. Activation was observed independent of whether CD46 was used as a cellular receptor and did not correlate with CD46 down-regulation. The virus itself was an activator of the alternative pathway and was covered by C3b/C3bi, resulting in some loss in infectivity without loss of virus binding to target cells. The cell surface expression of MV fusion (F), but not haemagglutinin, envelope protein resulted in complement activation of the Factor B-dependent alternative pathway in a dose-dependent manner and F–C3b complexes were formed. The underlying activation mechanism was not related to any decrease in cell surface expression of the complement regulators CD46 and CD55. The C3b/C3bi coating of MV-infected cells and virus should ensure enhanced targeting of MV antigens to the immune system, through binding to complement receptors.

The first three authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Measles virus (MV) is responsible for an acute childhood disease that, each year, infects almost 40 million infants and causes 0·8 million deaths (Stein et al., 2003). Natural infection by this virus is characterized by a profound cellular immunodepression favouring opportunistic secondary infections, but also the induction of efficient life-long humoral and cellular immunity (Auwaerter et al., 1999; Griffin et al., 1994; Hirsch et al., 1984). While immunization with an attenuated MV strain provides efficient protection, the use of inactivated MV vaccine in the early sixties resulted in the induction of an exacerbated atypical measles disease after secondary natural infection (Fulginiti et al., 1967). The immunodepression is characterized by ablation of delayed-type hypersensitivity, impaired in vitro lymphocyte proliferation and a shift towards a Th2 response (Auwaerter et al., 1999; Griffin et al., 1994). Thus, MV infection represents a challenging model to understand how a virus can simultaneously immunocompromise its host and induce a strong virus-specific immunity.

The recently recognized key role of the innate immunity in the induction of cognate immunity (see Aderem & Ulevitch, 2000; Fearon, 2000, for reviews) led us to explore further the possible contribution of complement in MV infection. Sissons and co-workers observed that MV Edmonston strain-infected human HeLa cells activate, in the absence of antibodies, the alternative pathway of human complement and that these cells were more sensitive to complement-mediated lysis (Sissons et al., 1979, 1980). To enter HeLa cells, MV uses the protein CD46 as a cellular receptor (Dörig et al., 1993; Naniche et al., 1993a). CD46 is a regulator of complement activation that acts as a cofactor of the soluble serine protease, Factor I, to cleave and inactivate covalently bound cell surface C3b and C4b into C3bi and C4bi (see Liszewski et al., 1991, for review). CD46 acts preferentially on the alternative pathway (Kojima et al., 1993) by inhibiting the amplification loop of C3b deposition (Devaux et al., 1999). During MV infection, CD46 is down-regulated because interaction with the MV haemagglutinin (H) protein results in enhanced internalization (Krantic et al., 1995; Naniche et al., 1993b) and/or intracellular retention of CD46 (Yant et al., 1997). This down-regulation was proposed to be responsible for the increased sensitivity of MV-infected cells to complement-mediated cell lysis (Schneider-Schaulies et al., 1995; Schnorr et al., 1995). However, only a small amount of CD46 can prevent human C3b deposition (Christiansen et al., 2000a) and residual levels of CD46 after infection may prevent C3b deposition.

CD46 is now generally considered to function as a receptor for vaccine and laboratory-adapted strains of MV only. Instead, all strains, including wild-type MVs, use CD150 (signalling lymphocytic activation molecule, or SLAM) as a cellular receptor (Tatsuo et al., 2000b). Accordingly, MV strains are referred as CD46non-using or CD46using.

We have attempted to clarify further the molecular mechanisms of alternative complement pathway activation by MV, whichever CD46 and/or CD150 cellular receptor it uses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antibodies, virus, cells and serum.
The following antibodies were used: mouse anti-CD46 monoclonal antibody (mAb) MCI20.6 and 10/88 directed against CD46 short consensus repeat (SCR) 1 and SCR3/4, respectively (Buchholz et al., 1997); mouse anti-CD55 12A12 antibody (Lozahic et al., 2000); simian polyclonal anti-MV-BIL BMS-94 serum; rabbit polyclonal antibody against the cytoplasmic tail of MV fusion (F) protein; anti-MV H mAb cl55; anti-MV F protein Y503; anti-CD150 mAb (Pharmingen); mAbs against C3b, iC3b and C3c (WM1; ECACC no. 92021211), the latter of which recognizes the C3 {beta}-chain (unpublished data); and phycoerythrin (PE)-labelled anti-mouse IgG(H+L) and anti-human IgG(H+L) (Beckman-Coulter).

Human serum (HS), isolated from ice-clotted blood of a single donor and human Factor B-depleted serum (Quidel) were stored as aliquots at –70 °C before use. IgG-depleted HS was obtained by incubating HS at 4 °C with an equal volume of protein G–agarose beads for 1 h just prior to the experiment. IgG-depleted HS was essentially non-reactive with CHO.CD46 cells and showed only residual labelling of MV-infected cells by flow cytometry. The residual anti-MV antibodies were devoid of significant neutralizing activity (data not shown). Heat-inactivated HS (30 min at 56 °C) was also used.

CHO (Chinese hamster ovary) fibroblasts CHO.CD46 (Devaux et al., 1999), simian B95a and human 293T cells were grown in Dulbecco's modified essential medium (DMEM) supplemented with 6 % foetal calf serum (FCS), 10 µg gentamicin ml–1, non-essential amino acids and 10 µg adenosine, deoxyadenosine and thymidine ml–1.

The Hallé strain of MV (CD46using) was produced in African green monkey Vero or human HeLa cells and purified on a sucrose gradient (Naniche et al., 1993a). The Lys-1 (Fayolle et al., 1999) and Ma93F (Lecouturier et al., 1996) MV strains (CD46non-using) were amplified in simian B95a cells. CHO.CD46, B95a and 293T cells were infected with MV at an m.o.i. of 0·1 or 1 for 1 h, washed and incubated with 10 µg zD-phe-L-Phe-Gly tripeptide ml–1 to prevent syncytium formation (Richardson & Choppin, 1983). The cells were used 48 h after MV infection.

Expression of MV H and F protein.
293T cells were transfected with 2 µg (unless otherwise indicated) of plasmid using Lipofectamin and OPTIMEM reagents (Invitrogen). The eukaryotic expression vectors used were pSC6-T7, pCAG-CD150, pCX2N KAH, pCX2N EdH and pCXN F encoding T7 polymerase, CD150, H from the KA MV strain (CD46non-using) and H and F from the Edmonston MV strain (CD46using), respectively (Tatsuo et al., 2000a).

Complement activation on cells.
Forty-eight hours after MV infection or 24 h after transfection, cells were harvested, washed with DMEM plus 0·05 % sodium azide (containing no FCS), then incubated for 30 min at 37 °C with IgG-depleted HS in the presence of MgCl2/EGTA unless otherwise indicated. Complement activation was stopped by rapid dilution with cold DMEM plus 0·05 % sodium azideand centrifugation at 400 g for 5 min. Following additional washing, the cells were incubated in 50 µl of an appropriate dilution of WM1 mAb at 4 °C for 30 min. Cell infection and CD46 expression were detected using murine monoclonal anti-H or -F and anti-CD46 antibodies, respectively. Immunolabelling was measured after incubation with PE-labelled anti-mouse Ig(H+L) conjugate and flow cytofluorometry as detailed elsewhere (Naniche et al., 1993a). In some experiments, CHO or CHO.CD46 cells (2·5x105) pre-loaded with MV for 1 h at 37 °C were used. After two washes to eliminate unbound virus, cells were incubated at 37 °C for 30 min with MgCl2/EGTA-supplemented IgG-depleted HS. In one experiment, the unbound virus was left and the HS was added directly to the virus/cell mixture. In some experiments, results were expressed as mean fluorescence value in arbitrary units. Fluorescence background level after incubation with the conjugate without the primary antibody remained within the ±20 % range and was therefore not subtracted. When CD46, CD55, H and F expression levels were compared after MV infection or transfection with expression vectors encoding the MV glycoproteins, the results were normalized and expressed as % expression, calculated as follow: % expression=(observed expression–background)/(maximal expression–background)x100, where maximal expression=expression level (in mean fluorescent units) observed in untreated cells for CD46 and CD55 and in MV-infected cells for H and F protein and background=fluorescence level in the absence of primary antibody (conjugate control).

MV binding after complement activation on the virus.
Purified MV Hallé was incubated for 30 min at 37 °C with MgCl2/EGTA-supplemented IgG-depleted HS (heat-inactivated or not). Complement activation was stopped by heat inactivation at 56 °C for 30 min. The virus/complement mixture was then incubated with CHO.CD46 cells. After a 1 h incubation and subsequent washing, the cells were stained for C3b and MV binding. Heat treatment did not change the MV binding properties (not shown).

Immunoprecipitation.
Cells (107) were infected with MV (m.o.i.=1) for 24 h and incubated with HS to allow activation of the alternative pathway for 20 min. After two washes in cold DMEM (without FCS) and lysis in a CHAPS/Triton X-100 buffer (50 mM Tris, pH 8, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 10 g CHAPS l–1, 1 % Triton X-100 and protease inhibitors), a pre-clearing step was performed with 20 µl of an irrelevant antibody, 9E10 anti-myc and anti-mouse Ig antibodies bound to protein G–Sepharose beads. WM1 antibody (40 µg) was then added to the lysate. After precipitation using anti-mouse Ig antibodies bound to protein G–Sepharose beads, bound proteins were eluted from the beads by heating at 95 °C with 30 µl of reducing Laemmli buffer and separated by PAGE. F protein and C3b {beta}-chain were detected by Western blotting using a rabbit anti-F cytoplasmic tail polyclonal antiserum and WM1 mAb, respectively.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
MV infection induces cell surface activation of the alternative complement pathway
CHO.CD46 cells, infected or not with MV, were incubated with IgG-depleted HS supplemented with MgCl2/EGTA and C3b deposition was analysed. Infection of CHO.CD46 cells was revealed by incubation with anti-H (grey histogram) and anti-F (white histogram) antibodies (Fig. 1, compare a and d). As expected, MV infection induced a down-regulation of human CD46 (Fig. 1, compare b and e). MV infection of CHO.CD46 cells resulted in a large increase in the level of C3b deposition on the cell surface after incubation with IgG-depleted HS (Fig. 1, compare f and c). As a control, heat-inactivated HS did not result in C3b deposition on cells (data not shown).



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Fig. 1. Influence of MV infection on activation of the alternative complement pathway. CHO.CD46 cells infected (d–f) or not (a–c) with Hallé MV were incubated with IgG-depleted HS supplemented with MgCl2/EGTA at 37 °C. After 1 h, C3b deposition (c, f), MV H and MV F expression (a, d) and CD46 expression (b, e) were determined by flow cytometry after immunolabelling using anti-C3b (WM1), anti-H (cl55, grey histogram), anti-F (Y503, white histogram) and anti-CD46 (MCI20.6) mAbs, respectively. Cells (5x103) were analysed and the results expressed as mean fluorescence values in arbitrary units. A conjugate control experiment was also performed in (d–f), resulting in similar fluorescence levels as displayed in (a), and therefore was not include in the figure.

 
Influence of MV binding on the cell surface activation of the alternative pathway
MV binding was observed on both CHO (Fig. 2a, grey histogram) and CHO.CD46 cells (Fig. 2d, grey histogram) at low and high levels, respectively. On CHO cells, which express endogenous hamster C regulatory proteins unable to act on human complement (Devaux et al., 1999), the presence of MV bound to the cell surface did not affect the high deposition of C3b following alternative pathway activation in the presence of MgCl2/EGTA (Fig. 2b, compare grey and white histograms). In contrast, the efficient regulation of C3b deposition typically observed on the CHO.CD46 cell surface (Fig. 2e, white histogram) was abolished by the presence of pre-bound MV (Fig. 2e, grey histogram), suggesting that the function of CD46 was blocked by the presence of MV. If the excess virus was not washed away prior to incubation with IgG-depleted HS, the level of C3b deposition was decreased on both cell lines (Fig. 2, compare grey histograms of b and c, and e and f). One possible explanation is that the unbound virus could have reduced the amount of available soluble C3b by activating complement on its surface.



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Fig. 2. Activation of the alternative complement pathway by MV. (a–f) Influence of MV binding on activation of the alternative complement pathway. CHO (a–c) and CHO.CD46 (d–f) cells were incubated with (grey histogram) or without (white histogram) purified MV. After 1 h at 37 °C, IgG-depleted HS supplemented with MgCl2/EGTA was added to washed (b, e) or unwashed (c, f) cells. After 1 h, C3b deposition (b, c, e, f) and MV binding (a, d) were determined by flow cytometry after immunolabelling using WM1 anti-C3b mAb or BMS-94 anti-MV antibody, respectively. (g–h) Complement activation by MV. Purified MV produced on HeLa cells was incubated at 37 °C with IgG-depleted HS (grey histogram). Prior to the incubation with CHO.CD46 cells, the virus/serum mixture was inactivated at 56 °C for 30 min. As a control, cells were incubated with Ig-depleted HS without virus (white histogram). After 1 h, MV binding was determined by flow cytometry after immunolabelling using BMS-94 anti-MV antibody (g) or WM1 anti-C3b mAb (h). Cells (5x103) were analysed and the results expressed as mean fluorescence values in arbitrary units.

 
Activation of the alternative complement pathway by MV
After direct activation of the alternative pathway by purified MV using MgCl2/EGTA-supplemented and IgG-depleted HS, virus binding and C3b deposition were detected on CHO.CD46 cells using an anti-MV antibody (Fig. 2g, grey histogram) and anti-C3b antibody (Fig. 2h, grey histogram). Since no labelling with anti-C3b antibody was observed when the cells were incubated with the IgG-depleted HS without virus (Fig. 2h, white histogram), this indicated that C3b deposition had occurred at the virus surface, prior to its binding to the cells. It did not affect MV binding to CD46, since the MV binding level detected by anti-MV antibodies was similar to that observed with untreated MV (data not shown). C3b bound to MV is unlikely to contribute significantly to the binding of MV to CD46. Indeed, stable C3b binding to CD46 occurs only in soluble form and at low salt concentration (Seya & Atkinson, 1989) or when C3b is covalently bound to the same membrane that harbours CD46 (i.e. it has only intrinsic activity; Oglesby et al., 1992). Binding of purified C3b dimers can be observed (Karp et al., 1996), but in the presence of whole serum, surface-bound C3b is quickly degraded into C3bi (unpublished data).

Activation of the alternative pathway by both CD46using and CD46non-using MV strains in the presence of a truncated CD46 proficient for complement regulation but unable to bind MV
Simian B95a cells were chosen as target cells because they express truncated simian CD46 molecules devoid of SCR1. Indeed, SCR1-deleted CD46 is unable to bind to MV, but efficiently regulates complement activation (Erlenhoefer et al., 2001; Murakami et al., 1998). B95a cells also express the simian CD150 (Tatsuo et al., 2000b), which enables infection by both CD46using and CD46non-using MV strains. As expected from the species cross-functionality of simian CD46 with human complement (Murakami et al., 1998), uninfected B95a cells did not exhibit C3b deposition after incubation with HS supplemented with MgCl2/EGTA (Fig. 3a). However, infection with either MV Hallé (CD46using) or Lys-1 strains (CD46non-using) resulted in a marked C3b deposition (Fig. 3e and i) without any down-regulation of CD46 (Fig. 3f and j). In contrast, CD150 was down-regulated after Lys-1 or Hallé infection (Fig. 3g and k). In this experiment, the CD150 down-regulation induced by MV Hallé was lower than that induced by MV Lys due to the lower level of H expression (Fig. 3, compare h and l). We therefore speculated that the complement activation was a consequence of the expression of the MV H and/or F envelope glycoproteins.



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Fig. 3. Lack of correlation between activation of the alternative pathway, CD46 down-regulation and its use as a cellular receptor. Simian B95a cells were infected (e–l) or not (a–d), with CD46using MV Hallé (e–h) or CD46non-using Lys-1 (i–l) strains for 48 h, incubated with HS supplemented with MgCl2/EGTA for 20 min at 37 °C and tested for cell surface deposition of C3b (a, e, i) and expression of CD46 (b, f, j), CD150 (c, g, k) and MV H protein (d, h, l) using WM1, CD46 SCR3/4-specific 10/88, anti-CD150 and cl55 mAbs, respectively. Conjugate control experiments are shown as black histograms in (a), (e) and (i).

 
Expression of MV F glycoprotein results in cell surface activation of the Factor B-dependent alternative pathway
The transient expression of the MV H protein from the KA (CD46non-using) strain on 293T cells resulted neither in an enhanced C3b deposition nor in down-regulation of cell surface CD46 when compared with the unrelated control plasmid (Fig. 4a). However, expression of the H protein from the Edmonston strain (CD46using) resulted in a significant CD46 down-regulation and a modest increase in C3b deposition. In contrast, the expression of the F protein from Edmonston strain alone induced strong C3b deposition, without affecting the expression of either of the complement regulators CD46 and CD55. The co-expression of F and H (KAH+F) also resulted into a strong C3b deposition. As expected, CD46using MV-infected cells also showed elevated C3b deposition and CD46 down-regulation. The level of C3b deposition appeared to parallel that of the level of F expression. Indeed, when 293T cells were transfected with various amounts of pCX2N-F, a linear relationship between the level of C3b deposition and F expression was observed (Fig. 4b; correlation factor r=0·954, 2{alpha}<0·001). No such correlation was observed between C3b deposition level and H or CD150 expression levels (Fig. 4b) indicating that only F results in complement activation. To ascertain that, in our experimental conditions, we were dealing with activation of the alternative pathway, 293T cells expressing the MV F glycoprotein were incubated with Factor B-depleted serum supplemented with EGTA/MgCl2. As expected, no C3b deposition could be observed (Fig. 5c), although the serum was still active in the classical pathway as shown by the high C3b deposition on 293T cells expressing the MV H glycoprotein and incubated in the presence of anti-H antibodies (Fig. 5g). We then investigated whether C3b binding to F occurs. Indeed, when cell surface-bound C3b from MV-infected cells was immunoprecipitated by WM1 antibody, some mature F1, but not immature F0, protein was found in the immunoprecipitate (Fig. 6). The co-immunoprecipitation of F with C3b was specific, since no F protein was found in the WM1 mAb immunoprecipitate from infected cells not incubated with MgCl2/EGTA-supplemented HS. This experiment was repeated three times with similar results. The lack of change in the apparent molecular mass of the co-immunoprecipitated F1 subunit suggested that the C3b {beta}-chain was covalently linked to the F2 subunit, which we could not detect as we did not have a suitable antibody. Using non-reducing PAGE, we failed to detect any high molecular mass complexes (>250 kDa), which would consist of the disulfide-bound C3b {alpha}- and {beta}-chains covalently linked to the disulfide-bound F1 and F2 subunits, most likely because of the poor electrotransfer efficiency of such huge complexes.



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Fig. 4. The MV F glycoprotein induces complement activation on human cells. (a) Human 293T cells were either infected with CD46using MV Hallé or transfected with a eukaryotic expression vector encoding MV H from CD46using MV (EdH) or CD46non-using MV (KAH), or MV F. After incubation with HS for 20 min at 37 °C in the presence of EDTA/MgCl2, the cell surface deposition of C3b (left histograms) and expression of CD46, CD55 and MV H and F proteins (right bar charts) were determined using WM1, CD46 SCR1-specific MCI20.6, 12A12, cl55 and Y503 mAbs, respectively. Conjugate control experiments are shown as black histograms. The small shift to the right of the conjugate control after transfection with pCX2N-F was not reproduced and did not influence the fluorescence level observed after anti-C3b labelling. (b) Dose-dependent relationship between the level of cell surface C3b deposition and F expression. 293T cells were transfected with various amounts (0·01–2 µg) of pCX2N-F, pCX2N-KAH or pCAG-CD150 and tested for their ability to activate the alternative pathway. The correlation factor r values for F, CD150 and H are 0·954, 0·465 and 0·423, respectively.

 


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Fig. 5. MV F glycoprotein expression results in activation of the Factor B-dependent alternative pathway. 293T cells were transfected with 2 µg pCX2N-F (a–d) or pCX2N-KAH (e–h) and tested for their ability to activate Factor B-depleted (c, g) or normal (d, h) HS in the presence of EGTA/MgCl2 (c, d) or anti-H antibodies (g, h). F (b) and H (f) protein expression and C3b deposition levels (c, d, g, h) were determined by analysing 5x103 cells by flow cytometry.

 


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Fig. 6. Co-immunoprecipitation of MV F protein and C3b after complement activation. MV-infected HeLa cells incubated (lanes 1 and 3) or not (lanes 2, 4 and 5) with HS were lysed and immunoprecipitated (lanes 1–4) with WM1 anti-C3b antibody and anti-Ig bound to protein G–Sepharose. Lane 5 shows the positions of the F0 and F1 proteins run in the same gel as lanes 3 and 4. The immunoprecipitate was analysed by Western blotting for the presence of C3b {beta}-chain (70 kDa) (lanes 1 and 2) and F0 (60 kDa) or F1 (40 kDa) proteins (lanes 3–5) using WM1 and rabbit polyclonal anti-F cytoplasmic tail antibodies, respectively. Note the presence of WM1 mouse antibody heavy (~55 kDa) and light chains (~25 kDa) in the immunoprecipitate revealed after incubation of WM1 and peroxidase anti-mouse Ig conjugate. Apparent molecular masses from lanes 1 and 2 and from lanes 3–5 cannot be directly compared because of the different running conditions during PAGE.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The envelope F protein of MV has been demonstrated here to induce the cell surface activation of the Factor B-dependent alternative pathway of human complement with massive C3b deposition on both virus and infected cells. This observation was common to both wild-type and attenuated vaccine MV strains and appeared to be independent of receptor usage (i.e. CD46 versus CD150). Direct activation of human complement has been observed on other human pathogenic enveloped viruses such as human immunodeficiency virus (HIV), cytomegalovirus (CMV) and Epstein–Barr virus (EBV). HIV and CMV induce antibody-independent activation of the classical pathway mediated by direct binding of the C1q molecule to gp160 of HIV (Dierich et al., 1993; Ebenbichler et al., 1991; Solder et al., 1989; Thieblemont et al., 1993) or to CMV-infected cells (Spiller & Morgan, 1998). Furthermore, EBV gp350 expressed on EBV and EBV-infected cells activates the alternative pathway of complement in the presence of specific IgG (Caudwell et al., 1990; Mold et al., 1988).

How MV F protein expression results in cell surface activation of the alternative pathway is unknown. The involvement of F-specific antibodies is unlikely because similar C3b deposition was observed with HS and IgG-depleted HS (data not shown) and it would imply that F, but not H, glycoprotein elicited antibodies able to prime for complement activation. Moreover, activation through the lectin pathway is also unlikely, because, like the classical pathway, it strictly requires the presence of Ca2+ ions (Thielens et al., 2001; Turner, 1998), which were absent in our complement activation assay carried out in the presence of MgCl2/EGTA. C3b deposition on the cell surface results from the activation of the intramolecular thioester bond of the C3b {alpha}-chain and the covalent attachment to nearby molecules (Sahu & Lambris, 2001). The finding of F1 protein co-immunoprecipitated with the C3b {beta}-chain indicates that during exposure to HS, some disulfide-bridged C3b {alpha}- and {beta}-chains may become covalently linked to cell surface F1 protein. As expected, the immature F0 protein, which is less prone to be expressed at the cell surface, was not co-immunoprecipitated. Whether the formation of the C3b–F complex is the primary event, which engages the uncontrolled activation of the alternative pathway, can be questioned. It seems unlikely, since the amount of F1 protein co-immunoprecipitated with the C3b {beta}-chain was low, in contrast with what one would have expected from a direct relationship between the level of F expression and the level of C3b deposition. Furthermore, several attempts to co-immunoprecipitate C3b using anti-F antibodies have failed. C3b displays a high degree of specificity in reacting with targets with a preference for some primary OH groups on serine and threonine residues (see Sahu & Lambris, 2001, for review). It is possible that F is substituted with oligosaccharides favouring the attachment of C3b. Historically, cell surfaces have been distinguished as being good or bad complement activators. However, the more recent discovery of the ubiquitous cell surface expression of strong complement activation regulators such as CD46 and CD55 has led to consideration that the activation properties of a cell surface are directly linked to the presence or absence of such regulators. CD46, CD55 and functionally related molecules, such as the mouse Crry1 protein, are partially species specific and, for example, mouse or hamster CD55 molecules efficiently control homologous complement but not human complement (Harris et al., 2000). Moreover, when cells are incubated with blocking anti-CD46 antibodies, they become activators of the alternative pathway of human complement (Devaux et al., 1999). Furthermore, the knock-down of Crry1 expression in mice results in abortion with a C3-mediated complement destruction of the foetus (Xu et al., 2000). Since the alternative pathway was activated on F protein-expressing cells in the presence of normal amounts of CD46 and CD55, we have to speculate how F protein results in the ‘escape’ of C3b from its two known regulators, CD46 and CD55. We did not observe any decrease in cell surface expression of either CD46 or CD55. A spatial sequestration of F protein (and C3b) outside CD55- and CD46-rich membrane areas is possible. It is known that, while F protein localizes in areas rich in cholesterol and glycosphingolipids, or rafts, CD46 does not (Manie et al., 2000; Vincent et al., 2000). But the glycosylphosphatidylinositol-anchored CD55 molecule is a constituent of rafts and fully able, when expressed alone, to control the amplification loop of the C3b deposition (Christiansen et al., 2000b).

When the infecting virus expresses CD46using H protein, this seems to partially interfere with CD46 function, as shown by the modest increase in C3b deposition observed when expressing isolated H protein from Edmonston CD46using MV strain compared with that observed with CD46non-using KA strain H protein. Two mechanisms may contribute to this: the down-regulation of CD46 and competition between H protein and C3b for binding to CD46. The down-regulation is unlikely to be sufficient to explain the loss of C3b deposition control, since CHO cell clones expressing comparable low amounts of CD46 are fully able to prevent the C3b amplification loop (Christiansen et al., 2000a). Although the SCR2 domain of CD46 is involved in the cofactor activity (Adams et al., 1991; Manchester et al., 1995) and the binding to MV H protein (Buchholz et al., 1996, 1997; Iwata et al., 1995; Manchester et al., 1995), the two functions map to distinct sites (Christiansen et al., 2000a; Liszewski et al., 2000).

What could be the role of the C3b deposited at the virus surface? IgG-depleted HS supplemented with MgCl2/EGTA reduced virus infectivity down to 20–25 % of that observed with heat-inactivated IgG-depleted HS (unpublished data) suggesting that complement activation results in partial virus inactivation. Such an inactivation occurring in vivo should hamper virus spreading throughout the body by free virus particles and favours cell-to-cell virus spreading. An antigen coupled to C3d displays a 1000–10 000-fold enhancement in immunogenicity (Dempsey et al., 1996). Thus, MV coating with C3b/C3bi and its natural C3d proteolytic derivative most likely contributes to the induction of a life-long-lasting, virus-specific immune response in a host who is paradoxically immunodepressed by the virus infection (Fugier-Vivier et al., 1997; Hicks et al., 1977; Karp, 1999; Yamanouchi et al., 1981).

In summary, we have shown that MV activates the alternative complement pathway with some neutralizing effect. This could play an important role, in vivo, to slow virus propagation by inactivating circulating virus and eliminating MV-infected cells, and to enhance the induction of a specific immune response by opsonizing MV antigens. A critical role of C3 in the pathology induced by gammaherpesviruses has recently been demonstrated (Kapadia et al., 2002). We propose that the activation of the alternative pathway is a primary mechanism of defence against MV infection, before the production of neutralizing anti-MV antibodies by the immune system.


   ACKNOWLEDGEMENTS
 
The authors thank B. Loveland, T. F. Wild, Y. Yanagi, M. Billeter, R. Fernandez-Munoz and A. Osterhaus for providing us with reagents. E. Fromm is acknowledged for her contribution to some data. This work has gained benefit from the use of the CeCIL facilities and apparatus. This work was supported in part by grants from CNRS (ATIPE, D. G.) and MENESR (PRFMMIP). P. D. was supported by a fellowship from the Fondation Marcel Mérieux. D. C. was supported by a Marie Curie EU and Fondation pour la Recherche Médicale fellowships. S. P. was supported by the Délégation Générale pour l'Armement.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 December 2003; accepted 27 January 2004.



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