A CD46CD[55–46] chimeric receptor, eight short consensus repeats long, acts as an inhibitor of both CD46 (MCP)- and CD150 (SLAM)-mediated cell–cell fusion induced by CD46-using measles virus

Dale Christiansen1, Emmanuel R. De Sousa1, Bruce Loveland2, Peter Kyriakou2, Marc Lanteri2, Fabian T. Wild3 and Denis Gerlierb,1

Immunité et Infections Virales, VPV, CNRS–UCBL UMR 5537, Faculté de Médecine Lyon–RTH Laennec, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France1
The Austin Research Institute, Heidelberg, Victoria 3084, Australia2
INSERM U404, CERVI, 69365 Lyon Cedex 07, France3

Author for correspondence: Denis Gerlier. Fax +33 4 78 77 87 54. e-mail gerlier{at}laennec.univ-lyon1.fr


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
According to their cellular receptor use, measles virus (MV) strains can be separated into two phenotypes, CD46-using and CD46-non-using. A long chimeric receptor, CD46CD[55–46], was generated from the CD46 backbone, encompassing the four short consensus repeat (SCR) domains of CD46 linked via a flexible glycine hinge to SCR1 and SCR2 of CD55, SCR3 and SCR4 of CD46 and the STP, transmembrane and cytoplasmic tail of CD46. This chimeric receptor was proficient for MV binding but deficient in mediating MV-induced cell-to-cell fusion and virus replication, possibly due to the extended distance between the MV haemagglutinin (H) binding site (CD46 SCR1–SCR2) and the cell membrane. When coexpressed with either wild-type CD46 or CD150, this fusion-incompetent receptor exerted a dominant negative effect and inhibited both cell-to-cell fusion and entry of MV with CD46-using, but not CD46-non-using, phenotype. A soluble octameric CD46–C4bp{alpha} exhibited similar CD46- and CD150-mediated fusion inhibition properties only against CD46-using MV. This suggests that the long CD46CD[55–46] receptor acts by sequestering incoming MV prior to its binding to the shorter functional CD46 or CD150 receptor.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
pH-independent fusion at the plasma membrane is characteristic of several viruses including retroviruses and paramyxoviruses. In measles virus (MV), both of the envelope proteins, haemagglutinin (H) and fusion (F) proteins, are required for fusion. They are organized as tightly associated complexes of H tetramers and F trimers (Lamb et al., 1999 ; Malvoisin & Wild, 1993 ; Plemper et al., 2000 ). The H protein binds to the receptor on the target cell (Devaux et al., 1996 ; Tanaka et al., 1998 ; Tatsuo et al., 2000a ) and enables the F protein to mediate the fusion process (Cattaneo & Rose, 1993 ; Tatsuo et al., 2000a ; Wild et al., 1991 ), probably through conformational changes required for fusion (Chen et al., 2001 ).

To date, two cell surface proteins have been identified that act as cellular receptors mediating MV entry, CD46 (or Membrane Cofactor Protein, MCP) (Dörig et al., 1993 ; Naniche et al., 1993 ) and CD150 (or Signalling Lymphocytic Activation Molecule, SLAM) (Erlenhoefer et al., 2001 ; Hsu et al., 2001 ; Tatsuo et al., 2000b ).

CD46 allows the entry of MV laboratory strains maintained in epithelial and fibroblastic cell lines, including all attenuated virus strains used as efficient human vaccines (Escoffier & Gerlier, 1999 ; Hsu et al., 1998 ; Kobune et al., 1990 ; Lecouturier et al., 1996 ; Parks et al., 2001 ; Schneider-Schaulies et al., 1995b ; Tanaka et al., 1998 ). However, CD46 is not used by recent MV isolates grown in simian B95 B cells (Hsu et al., 1998 ; Kobune et al., 1990; Lecouturier et al., 1996 ; Murikami et al., 1999 ), or from throat swabs from infected individuals (Ono et al., 2001b ). Whether some natural wild-type strains can use CD46 has so far been obscured by their in vitro isolation in cell lines expressing CD46 but not CD150, or in B95 cells, which express CD150 but very little full-length functional CD46 (Hsu et al., 1998 ; Manchester et al., 2000 ; Murikami et al., 1999 ). Thus, depending on the cell type used for virus growth, MV strains use either CD150 or both CD46 and CD150; these virus strains will be referred as CD46-non-using MV and CD46-using MV, respectively. The ability of MV H to bind to CD46 has been mapped to critical residues at positions 211, 451 and 481 of the H protein (Bartz et al., 1996 ; Hsu et al., 1998 ; Lecouturier et al., 1996 ).

CD46 is a regulator of complement activation and is expressed on all human nucleated cells. CD46 is a type I transmembrane glycoprotein, which contains four short consensus repeat (SCR) domains. The MV H binding site has been mapped lying on the top face of the molecule contacting the two N-terminal SCR1–SCR2 domains (Buchholz et al., 1997 ; Iwata et al., 1995 ; Manchester et al., 1995 , 1997 ; Mumenthaler et al., 1997 ), as confirmed by their 3-D structure (Casanovas et al., 1999 ) (Fig. 1). CD46 usage by MV strains correlates with the ability to induce CD46 down-regulation at the cell surface (Lecouturier et al., 1996 ; Schneider-Schaulies et al., 1995a , b ).



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Fig. 1. Schematic drawing of potential MV receptors used in this study. STP and proximal regions of CD46 are drawn as quadrilateral structures and stars indicate the relevant binding site for the MV H protein.

 
CD150 was recently identified as another MV receptor allowing the entry of all MV strains tested so far (Erlenhoefer et al., 2001 ; Hsu et al., 2001 ; Ono et al., 2001a ; Tatsuo et al., 2000b ). CD150 is a type I transmembrane glycoprotein that is expressed on thymocytes, CD45Rohigh memory T cells and some B cells. It is rapidly induced on T, B and dendritic cells after activation (see Tatsuo et al., 2000b , for review). It belongs to the CD2 family with a short ectodomain made of two Immunoglobulin(Ig)-like V and C2 type domains. The binding site for MV H has been located on the N-terminal V domain (Ono et al., 2001a ) (Fig. 1). After MV infection, CD150 is down-regulated (Erlenhoefer et al., 2001 ).

A previous study has described the first (and so far only) example of a virus receptor with dominant negative properties towards the fusion step allowing the entry of an enveloped virus. Indeed, a long CD46/CD4 chimera was found to bind MV and inhibit the MV-induced cell-to-cell fusion induced by a short CD46/CD4 chimera (Buchholz et al., 1996 ). Here we report the construction of another CD46-based receptor, which displays fusion inhibitory activity against both of the wild-type CD46 and CD150-mediated MV entry mechanisms.


   Methods
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Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus, cell lines, antibodies and reagents.
The Hallé MV strain, which uses CD46 as a cellular receptor (Naniche et al., 1993 ), was propagated in Vero cells, and the Ma93F and Lys-1 MV strains, which cannot use CD46 (Fayolle et al., 1999 ; Lecouturier et al., 1996 ; this paper), were propagated in B95 cells. The following cells lines were used and maintained in Dulbecco’s modified minimum essential medium supplemented with 6% foetal calf serum, gentamycin, L-glutamine and 10 mM HEPES: CHO (Chinese hamster ovary) and CHO.CD46 (Christiansen et al., 2000b ) cells, Epstein–Barr virus (EBV)-transformed simian B95a B lymphoblastoid cells, and human 293-EBNA fibroblastic cells expressing the EBV nuclear antigen (Christiansen et al., 2000a ). The following antibodies were used: anti-CD150, A12 (Pharmingen Becton–Dickinson) anti-CD46, MCI20.6 recognizing the MV H binding site on SCR1 (Buchholz et al., 1997 ; Naniche et al., 1993 ), GB24 recognizing SCR4 (and SCR3) (Adams et al., 1991 ; Christiansen et al., 2000c ) and 11C7 recognizing SCR1–SCR2 (Christiansen et al., 2000c ), anti-CD55 recognizing SCR1–SCR2 12A12 (Christiansen et al., 2000c ), anti-H cl55 recognizing MV H glycoprotein (Giraudon & Wild, 1985 ) and anti-F Y503 recognizing MV F glycoprotein (Christiansen et al., 2000a ). The octameric soluble sCD46–C4bp{alpha} protein has been described elsewhere (Christiansen et al., 2000a ).

{blacksquare} Generation of chimeric CD46CD[55–46] protein and cell transfectants.
The wild-type cell-surface CD46 isoform used in this study includes STP BC and cytoplasmic tail 1. The chimeric CD46CD[55–46] protein included the insertion between CD46 SCR4 and STP B of a flexible glycine hinge together with four SCR domains: SCR1 and SCR2 from CD55, and SCR3 and SCR4 from CD46 sequence (Fig. 1). It was generated using splice overlap extension–polymerase chain reaction (SOE–PCR) (Horton et al., 1989 ), confirmed by sequencing and expressed after ligation of the cDNA into the end-filled Xba1 site of the APEX3p vector, which contains the EBV Ori (Christiansen et al., 2000b ). The flanking amino acid sequence of the CD46[SCR4] to CD55[SCR1] junction (CD46 sequence in bold, glycine hinge underlined, CD55 sequence in italics) was CLKV:GGGKG:DCGL, and the CD55[SCR2] to CD46[SCR3] junction was FCKK:VLCT. Stable CHO–K1 fibroblasts expressing the chimeric protein were derived by transfection, selection with puromycin and cloning as previously described (Christiansen et al., 2000c ). Transient CD46CD[55–46]-expressing cells were derived from B95 and 293-EBNA cells transfected with APEX3p–CD46CD[55–46] plasmid using the lipofectamine reagent (Invitrogen). The expression of the EBNA protein, in both cell lines, ensured the episomal replication of the APEX3p vector and high expression of the CD46CD[55–46] protein in most if not all cells. One day after transfection, over 90% of 293-EBNA cells expressed CD46CD[55–46] molecules and they were used for the infection test with MV, or as the ‘receptor’ cell partner in fusion assays. The B95–CD46CD[55–46] cells were first selected by growth in the presence of puromycin for 10–14 days before use and more than 95% of cells expressed the chimeric receptor.

{blacksquare} Protein expression assays.
The expression of CD46, CD46CD[55–46] and MV H glycoprotein were measured at the cell surface after incubation with appropriate antibodies, labelling with phycoerythrin anti-mouse Ig and flow cytometry as detailed previously (Naniche et al., 1993 ). The expression was also tested by Western blot after SDS–PAGE separation under non-reducing conditions and use of 12A12 and 11C7 antibodies as probes according to Manié et al. (2000 ).

{blacksquare} Virus binding and cell fusion assays.
The MV binding assay has been described previously in detail (Naniche et al., 1993 ). The fusion properties of the chimeric protein were determined by visual assessment of syncytia formation and/or by the use of a virus-based quantitative cell fusion-dependent reporter gene system as detailed previously (Christiansen et al., 2000a ). For inhibition studies with sCD46–C4bp{alpha}, the ‘fusing’ partners (105 cells in 100 µl) were incubated with 100 µg/ml of sCD46–C4bp{alpha} for 30 min at 4 °C prior to mixing with the ‘receptor’ partners. The results were expressed as percentage fusion inhibition.

{blacksquare} Virus infectivity determination.
The cells were infected with MV (m.o.i.=1) for 2 h at 37 °C. Non-adsorbed virus was removed and cells were incubated overnight at 37 °C in complete culture medium. One or 2 days post-infection (p.i.), the percentage of cells expressing the MV H protein was determined by flow cytometry, as described above.

{blacksquare} sCD46–C4bp{alpha} binding assay.
The binding of 10 µg/ml of sCD46–C4bp{alpha} to 2x105 MV-infected B95 cells was performed as detailed previously (Christiansen et al., 2000a ). The results were expressed as percentage binding to MV Hallé-infected B95 cells as a function of H expression level measured in parallel by cytometry using anti-H cl55 antibody.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Ability of CD46CD[55–46] to bind MV
The CD46CD[55–46] protein was found to be stably expressed on the cell surface of CHO cells as shown by its reactivity with both the anti-CD46 (Fig. 2, Table 1) and anti-CD55 antibodies (Table 1). CHO.CD46CD[55–46] cells were able to bind a much higher amount of MV than CHO cells (Fig. 2). Comparison of MV-binding ability of CHO.CD46 and CHO.CD46CD[55–46] cells revealed that the latter, which expressed a lower amount of virus binding site as detected by labelling with the anti-SCR1 MCI20.6 antibody, was more efficient than the former. Such an increase in MV-binding ability of an elongated receptor is in agreement with a previous finding using a CD46/CD4 chimeric receptor (Buchholz et al., 1996 ; Devaux et al., 1997 ). In contrast and as expected, the expression of CD46 or CD46CD[55–46] protein did not result in a significantly enhanced binding of CD46-non-using MV Ma93F strain. B95 cells, which expressed CD150 and a truncated SCR1- CD46 (Erlenhoefer et al., 2001 ; Hsu et al., 1998 ; Iwata et al., 1995 ; Murikami et al., 1998 ), bound CD46-using Hallé and CD46-non-using Ma93F MV with similar, albeit low, efficiency. This poor MV-binding ability of CD150 receptors was also noticed on human CD150-expressing rodent cell lines (unpublished data) and is in agreement with data reported by others (Tatsuo et al., 2000b ).



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Fig. 2. Ability of the CD46CD[55–46] chimera to bind to MV. CHO, CHO.CD46, CHO.CD46CD[55–46] and B95 cells were analysed for cell surface expression of receptor using MCI-20.6 anti-SCR1 antibody and for ability to bind CD46-using Hallé or CD46-non-using Ma93F MV strain revealed with MV H-specific antibody by flow cytometry.

 

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Table 1. Cell surface expression of MV cellular receptors and their ability to mediate MV-induced cell–cell fusion

 
Inability of CD46CD[55–46] to mediate MV-induced cell–cell fusion
When tested as a ‘receptor’ partner in a cell–cell fusion assay, the CD46CD[55–46] molecule was unable to mediate fusion with a cell partner expressing the MV H and F proteins derived from a MV strain able to bind to CD46 (CD46-using MV) (Table 1). As expected, cell–cell fusion was observed between CHO.CD46 cells and cells expressing MV H and F derived from a CD46-using MV strain, but not with CD46-non-using MV-infected cells. Likewise, B95 cells, which express CD150 and very little full-length CD46 but a large amount of SCR1- truncated CD46 (Erlenhoefer et al., 2001 ; Hsu et al., 1998 ; Iwata et al., 1995 ; Murikami et al., 1998 ), reacted with SCR3/4-specific GB24 but not with SCR1-specific MCI20.6 anti-CD46 antibody, and, as expected, were able to fuse with both CD46-non-using and CD46-using MV-infected cells.

Inability of CD46CD[55–46] to mediate MV infection
Stable expression of the CD46CD[55–46] protein was unable to mediate efficient MV replication as shown by the very low expression of MV H at the cell surface 48 h p.i. (Fig. 3), not significantly different to that observed on infected CHO cells. In contrast, MV H was expressed at a significant level after infection of CHO.CD46. Moreover, this infection induced the down-regulation of CD46, whereas the CD46CD[55–46] expression was slightly but not significantly increased.



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Fig. 3. Inability of the CD46CD[55–46] chimera to mediate MV infection of CHO cells. CHO, CHO.CD46 and CHO.CD46CD[55–46] were mock-infected or infected with CD46-using Hallé MV and tested after 48 h for cell surface expression of receptor and MV H by flow cytometry. +, MV-infected cells; -, mock-infected cells.

 
CD46CD[55–46] inhibits both CD46- and CD150-mediated infection by CD46-using MV
CD46CD[55–46] was co-expressed with either the CD46 or CD150 functional cellular receptor by transient expression in the CD46+ human fibroblastic cell line 293-EBNA or in the CD150+ simian B95 cells. The expression of CD46 and CD46CD[55–46] was verified by Western blot (Fig. 4) and by flow cytometry (Fig. 5a, c). The surface expression of the chimeric molecule on 293-EBNA cells was shown by a small shift to the right of the anti-CD46 labelling, which also recognized endogenous CD46 (Fig. 5a). After infection with the CD46-using MV Hallé strain, syncytia were readily observed with both 293 and B95 cells (Fig. 6c, g) but not when the cells expressed the CD46CD[55–46] molecule (Fig. 6d, h). In contrast, syncytia induced by the CD46-non-using MV Lys-1 strain was not inhibited when B95 cells expressed the CD46CD[55–46] molecule (Fig. 6, compare i and j). These results were confirmed by the strong reduction in H expression (~90%) on cells expressing the chimeric receptor after infection with CD46-using MV Hallé strain (Fig. 5b, d) but not after infection with the CD46-non-using MV Lys-1 strain (Fig. 5e). Taken together, these data reveal that the chimeric CD46CD[55–46] receptor has a dominant negative function as it can inhibit the infection of CD46+ 293-EBNA cells and CD150+ B95 cells by CD46-using MV strain.



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Fig. 4. Western blot of 293-EBNA cells (lanes 1, 2) and B95 (lanes 3, 4) transfected with Apex3p–CD46CD[55–46] (lanes 1, 3) or not (lanes 2, 4) and probed using anti-CD55 and anti-CD46 antibodies. CD46CD[55–46] and CD46 migrate in non-reducing conditions with apparent molecular masses of 85 and 66 kDa respectively.

 


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Fig. 5. Reduction in cell surface expression of H protein on transfected 293-EBNA (a, b) and B95 (c, d, e) cells induced by the expression of CD46CD[55–46] (a, c, light grey histograms) after infection with the CD46-using MV Hallé strain (b and d, respectively, light grey histograms), but not after infection with the CD46-non-using MV Lys-1 strain (c, light grey histograms). CD46 expression (a, c) and H expression (b, d, e) on control non-transfected cells are shown by dark grey histograms. Note that on in (e), the two histograms fully overlap.

 


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Fig. 6. Syncytium formation within 293-EBNA cells (a–d) and B95 cells (e–j) infected by CD46-using MV Hallé strain for 24 h (m.o.i.=1) (c, d, g, h) is inhibited upon expression of chimeric CD46CD[55–46] molecule (d, h) whereas syncytium formation within B95 cells infected by CD46-non-using MV Lys-1 strain (i, j) is not inhibited upon expression of CD46CD[55–46] (j). Magnification x10. n.i., Not infected.

 
Inhibition of MV-mediated cell–cell fusion using soluble sCD46–C4bp{alpha} and correlation with inhibition of MV infection by chimeric CD46CD[55–46] according to MV strain
To get an insight into the possible mechanism underlying the inhibitory effect of the chimeric CD46CD[55–46] receptor, its activity was compared with that of a soluble octameric form of CD46, sCD46–C4bp{alpha}, which we have previously reported to be a potent inhibitor of MV entry mediated by CD46 (Christiansen et al., 2000a ). As expected, sCD46–C4bp{alpha} bound to B95 cells infected by CD46-using MV Hallé strain but not by CD46-non-using MV Lys-1 and Ma93F strain (Fig. 7, white columns). As observed with the CD46CD[55–46] receptor, the soluble sCD46–C4bp{alpha} was able to strongly inhibit the B95 cell–cell fusion induced by the CD46-using MV Hallé strain, but had no significant effect on that induced by CD46-non-using MV Lys-1 and Ma93F strains (Fig. 7, grey columns). Thus, the transmembrane chimeric CD46CD[55–46] receptor and the soluble octameric CD46 seem to display a similar inhibitory phenotype on MV infection.



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Fig. 7. Correlation of the ability of soluble sCD46–C4bp{alpha} to bind to B95 MV-infected cells (white columns), to inhibit MV-induced B95 cell–cell fusion (grey columns) with the ability of the chimeric CD46CD[55–46] receptor to inhibit MV H expression (dark columns) in B95 cells depending on the infecting CD46-using (Hallé) or CD46-non-using (Lys-1 and Ma93F) MV strains. As a control, the inhibition of MV-induced B95 cell–cell fusion by anti-F (hatched columns) antibodies is shown.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
By analogy with the long CD46/CD4 protein composed of the four SCR domains of CD46 fused to the four Ig-like domains of the CD4 molecule (Buchholz et al., 1996 ), the CD46CD[55–46] chimeric protein was generated with eight SCRs with SCR1 and SCR2 of CD46, which harbour the functional MV binding site (Buchholz et al., 1997 ; Casanovas et al., 1999 ; Iwata et al., 1995 ; Manchester et al., 1995 , 1997 ; Mumenthaler et al., 1997 ), being located at the N-terminus. These two chimeric proteins are expected to have a similar size, ~22 nm for the long CD46/CD4 protein and ~20 nm for CD46CD[55–46], according to previous modelling and 3-D structures of two and four SCR lengths (Buchholz et al., 1996 ; Casanovas et al., 1999 ; Mumenthaler et al., 1997 ). The CD46CD[55–46] was unable to mediate the MV H- and F-induced cell-to-cell fusion. One could speculate that this is due to increased protein length and hence moving the MV binding site away from the membrane. In addition, this CD46CD[55–46] protein exerted a dominant negative effect in inhibiting the fusion mediated by MV H interaction with wild-type CD46. This result is in agreement with the inhibitory effect of the long CD46/CD4 receptor on the fusion mediated by a short CD46/CD4 chimeric protein made of CD46 SCR1–SCR2 and the fourth CD4 Ig-like domain (Buchholz et al., 1996 ).

To address the specificity of our observations, the activity of CD46CD[55–46] was also tested on MV entry mediated by the so far ‘universal’ MV receptor, CD150. Simian B95 cells were chosen because they express only a limited amount of functional CD46 receptor. Indeed, although an mRNA encoding a full-length simian CD46, with functional MV properties, has been recovered by RT–PCR, the major CD46 mRNA encoded a truncated non-functional SCR1- receptor (Hsu et al., 1998 ; Murikami et al., 1998 ). B95 cells are poorly (Hsu et al., 1998 ) or not labelled by anti-SCR1 antibodies (Erlenhoefer et al., 2001 ; this paper). Following MV infection with a CD46-using MV strain, no down-regulation of simian CD46 labelled with anti-SCR3 or 4 antibodies could be detected (Erlenhoefer et al., 2001 ; D. Gerlier, unpublished data). In addition, the binding of CD46-using and CD46-non-using MV strains to B95 cells is completely inhibited by anti-CD150 antibodies with similar efficiencies (Erlenhoefer et al., 2001 ). The cell-to-cell fusion and virus infection (measured by H expression) in B95 cells were both inhibited in the presence of CD46CD[55–46] receptor when infected with a CD46-using MV strain, but not with a CD46-non-using MV strain. Similar results were obtained using several other MV strains such as CD46-using MV Edmonston, recombinant Edtag (Radecke et al., 1995 ), Y15 (Giraudon et al., 1988 ), Y22 (Giraudon et al., 1988 ) and the CD46-non-using MV Ma93F strains (data not shown). This indicates that this chimeric receptor can inhibit MV infection probably by inhibiting the virus entry mediated by both of the CD46 and CD150 receptors provided that the virus expressed H glycoprotein is able to bind to this fusion-incompetent receptor. The potent inhibitory effect of the chimeric receptor on CD150-mediated MV infection correlates with the much lower binding efficiency of MV to CD150 than to CD46 receptors (see data with CD46-using MV Hallé in Fig. 2).

After infection with CD46-using MV, we noticed that while the syncytia formation was almost fully inhibited (see Fig. 6), the level of cell surface H expression was strongly but not completely inhibited. In particular, whereas ~82% of B95 cells expressed a significant level of CD46CD[55–46], up to ~52% expressed a detectable level of H (see Fig. 5). Similar results were obtained when the expression of the F glycoprotein was analysed (not shown). This suggests that cell–cell fusion is more sensitive to receptor-mediated inhibition than virus infection, as observed with other soluble virus entry inhibitors (Christiansen et al., 2000a ).

When studying the CD46/CD4 chimeric receptors, the dominant negative interference of a long fusion-incompetent receptor over a short functional receptor, even at an unfavourable molar ratio, argued for the existence of a MV fusion complex (Buchholz et al., 1996 ). In particular, one could speculate whether the presence of a fusion-incompetent long receptor among several functional homologous receptors involved in a single MV fusion complex can disable the whole molecular scaffold. The ability of a fusion-incompetent receptor made from the CD46 backbone to inhibit MV-induced fusion of the short structurally unrelated CD150 receptor made of two Ig-like domains, together with inhibitory activity of the soluble sCD46–C4bp{alpha}, suggest another inhibitory mechanism. A long receptor (estimated to be >20 nm) is likely to be more accessible from the outside of the cells than a short one such as CD46 (~10 nm) or CD150 (~6 nm), and can sequester any cell surface-contacting MV H glycoproteins. This interaction could lead to an irreversible conformational inactivation of the companion F molecule, as suggested by the potent MV inhibitory activity of the soluble sCD46–C4bp{alpha} (Christiansen et al., 2000a ). This effect would be amplified if a single CD46-based receptor could interact sequentially with several MV H–F complexes.

The efficient inhibitory effect of CD46CD[55–46] on the CD150-mediated MV infection and the higher MV-binding efficiency of CD46-based molecules suggests that, when a CD46-using MV strain infects human cells expressing both CD46 and CD150 receptors, i.e. activated B and T cells, dendritic cells and memory T cells, a competition for receptor usage may occur. Such competition could play a role in the attenuation process of live attenuated MV vaccine. Similarly, engineered H protein for retargeting to a new cellular receptor without abrogation of the binding to the natural receptor as recently described (Hammond et al., 2001 ; Schneider et al., 2000 ) may lead to competition for receptor usage.


   Acknowledgments
 
The technical contribution of Perrine Martin is acknowledged. The authors thank all the members of Immunité & Infections Virales team for helpful discussions. The authors thank M. Billeter and R. Fernandez-Munoz for providing various reagents. The following reagent was obtained through the AIDS Research and Reference Reagent program, Division of AIDS, NIAID: vCB21R–lacZ from Dr C. C. Broder, P. E. Kennedy and E. A. Berger. This work was supported in part by grants from CNRS, and the Ministère de l’Education Nationale et de la Recherche et de la Technologie (PRFMMIP) (D.G.) and the NHMRC (Australia) (B.L., P.K., M.L.). D.C. was supported by a Marie Curie EU Fellowship and a Fondation pour la Recherche Médicale Fellowship.


   Footnotes
 
b Wild-type CD46 and CD55 sequences in this paper refer to GenBank accession numbers A18585 and M30142, respectively.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 22 October 2001; accepted 4 January 2002.