Immunité et Infections Virales, VPV, CNRSUCBL UMR 5537, Faculté de Médecine LyonRTH 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
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Abstract |
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Introduction |
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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 SCR1SCR2 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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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.
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Methods |
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Generation of chimeric CD46CD[5546] 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[5546] 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 extensionpolymerase chain reaction (SOEPCR) (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 CHOK1 fibroblasts expressing the chimeric protein were derived by transfection, selection with puromycin and cloning as previously described (Christiansen et al., 2000c
). Transient CD46CD[5546]-expressing cells were derived from B95 and 293-EBNA cells transfected with APEX3pCD46CD[5546] 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[5546] protein in most if not all cells. One day after transfection, over 90% of 293-EBNA cells expressed CD46CD[5546] molecules and they were used for the infection test with MV, or as the receptor cell partner in fusion assays. The B95CD46CD[5546] cells were first selected by growth in the presence of puromycin for 1014 days before use and more than 95% of cells expressed the chimeric receptor.
Protein expression assays.
The expression of CD46, CD46CD[5546] 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 SDSPAGE separation under non-reducing conditions and use of 12A12 and 11C7 antibodies as probes according to Manié et al. (2000
).
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 sCD46C4bp
, the fusing partners (105 cells in 100 µl) were incubated with 100 µg/ml of sCD46C4bp
for 30 min at 4 °C prior to mixing with the receptor partners. The results were expressed as percentage fusion inhibition.
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.
sCD46C4bp
binding assay.
The binding of 10 µg/ml of sCD46C4bp 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.
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Results |
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Inability of CD46CD[5546] to mediate MV infection
Stable expression of the CD46CD[5546] 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[5546] expression was slightly but not significantly increased.
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Discussion |
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To address the specificity of our observations, the activity of CD46CD[5546] 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 RTPCR, 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[5546] 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[5546], 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 cellcell 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 sCD46C4bp
, 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 sCD46C4bp
(Christiansen et al., 2000a
). This effect would be amplified if a single CD46-based receptor could interact sequentially with several MV HF complexes.
The efficient inhibitory effect of CD46CD[5546] 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.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 October 2001;
accepted 4 January 2002.