Interaction of CD46 with measles virus: accessory role of CD46 short consensus repeat IV

Dale Christiansen1, Bruce Loveland2, Peter Kyriakou2, Marc Lanteri2, Carine Escoffier1 and Denis Gerlier1

Immunité et Infections Virales, IVMC, CNRS–UCBL UMR 5537, 69372 Lyon Cedex 08, France1
The Austin Research Institute, Heidelberg, Victoria 3084, Australia2

Author for correspondence: Dale Christiansen. Fax +33 4 78 77 87 54. e-mail christia{at}laennec.univ-lyon1.fr


   Abstract
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I–II–III–IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Measles virus (MV), a member of the order Mononegavirales,uses the human cell surface protein CD46 (or membrane cofactor protein) as a cellular receptor (Naniche et al., 1993a ). The envelope, composed of the haemagglutinin (H) and fusion (F) glycoproteins, surrounds the ribonucleoparticle containing the viral RNA and transcription–replication machinery. The H protein is responsible for virus binding to the cellular receptor, CD46, and the F protein subsequently induces fusion between the viral envelope and the cell membrane.

CD46 is a transmembrane glycoprotein that belongs to the regulators of complement activation (RCA) gene family (Liszewski et al., 1991 ). It protects host cells from autologous complement (Devaux et al., 1999 ; Seya et al., 1986 ). The dominant structural units of CD46 are the short consensus repeat (SCR) modules of 60–64 amino acids that are responsible for complement binding and regulatory functions. Variable RNA splicing generates multiple CD46 isoforms that differ in the heavily glycosylated serine–threonine–proline (STP)-rich regions (designated A, B and C) and cytoplasmic tail (designated 1 or 2). All of the common CD46 isoforms can serve as receptors for MV (Gerlier et al., 1994 ; Manchester et al., 1994 ).

MV binding to CD46 was initially shown to involve an interaction of the ectodomain of the envelope H glycoprotein (Devaux et al., 1996 ) and SCRs I and II of CD46 (Buchholz et al., 1997 ; Iwata et al., 1995 ; Manchester et al., 1995 ). The N-glycan of CD46 SCR II appears to be essential for maintaining a conformation of CD46 that can be recognized by MV (Maisner et al., 1996 ). The primary binding sites of the MV–CD46 interaction have been further characterized as covering the interface between the SCR I and II domains (Buchholz et al., 1997 ; Casasnovas et al., 1999 ; Hsu et al., 1997 ; Manchester et al., 1997 ; Mumenthaler et al., 1997 ). Furthermore, chimeric proteins generated between CD46 and CD4 revealed that the SCR III–IV domains of CD46 could modulate the binding of both MV and a recombinant soluble form of the H protein (Buchholz et al., 1997 ; Devaux et al., 1997 ).

Infection of target cells with various MV strains results in down-regulation of CD46 from the cell surface (Naniche et al., 1993b ; Schneider-Schaulies et al., 1995b , 1996 ). This down-regulation is linked to the contact between the H glycoprotein and CD46 (Krantic et al., 1995 ; Naniche et al., 1993b ) and, thus, can be uncoupled from virus fusion (Firsching et al., 1999 ). CD46 down-regulation has been proposed to enhance the sensitivity of infected cells to complement-mediated lysis (Schneider-Schaulies et al., 1996 ; Schnorr et al., 1995 ). All laboratory-adapted MV strains down-regulate CD46, whereas several recently isolated strains do not (Schneider-Schaulies et al., 1995a , b ). This inability to down-regulate CD46 is due to phenotypic differences between MV strains, and two amino acids in the H protein (Val-451 and Tyr-481) have been identified as being critical for receptor down-regulation (Bartz et al., 1996 ; Lecouturier et al., 1996 ). An additional novel site in MV H necessary for the high-affinity CD46–H interaction (amino acids 473–477) was also identified recently (Patterson et al., 1999 ). By using chimeric CD46–CD4 proteins, the CD46 SCR I and II domains, which contain the primary H-binding site, were shown to be necessary for CD46 down-regulation (Firsching et al., 1999 ). In addition, the cytoplasmic Tyr–X–X–Leu motif of CD46 was demonstrated to be essential for its down-regulation by the MV H protein in persistently infected cells (Hirano et al., 1996 ; Yant et al., 1997 ).


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Generation of chimeric CD46 isoforms and stable transfectants.
The wild-type cell-surface CD46 isoform used in this study includes STP BC and cytoplasmic tail 1 and is designated I–II–III–IV. The chimeric CD46/DAF proteins included the exchange of CD46 SCR III [i.e. CD46(SCR I+II)–DAF(SCR 3)–CD46(SCR IV)], designated x3DAF, and the exchange of CD46 SCR IV [CD46(SCR I+II+III)–DAF(SCR 4)], designated x4DAF. An additional control chimeric protein consisting of an exchange of CD46 SCR domains I and II [DAF(SCR 1+2)–CD46(SCR III+IV)], designated x1/2DAF, was also generated. In all chimeras, the remainder of the molecule includes STP BC and cytoplasmic tail 1. CD46/DAF chimeras were generated by using splice-overlap extension PCR (Horton et al., 1989 ). Each DNA insert was ligated into the end-filled XbaIsite of the APEX-3p vector. The constructs were transformed into Escherichia coli XL-1 Blue (Stratagene).

Cloning site integrity, orientation of the insert, presence of the desired SCR substitution and CD46/DAF SCR junctions were confirmed by sequencing. The flanking amino acid sequence of the x3DAF junction (CD46 sequence in bold, DAF sequence in italics) was ICEK:KSCP; x4DAF was ECKV:IYCP and x1/2DAF was FCKK:VLCT. For wild-type CD46 and DAF sequences, refer to GenBank accession numbers A18585 and M30142, respectively.

Stable CHO-K1 fibroblasts expressing the chimeric proteins were derived by transfection, selection with puromycin, single-cell sorting and cloning, as described previously (Christiansen et al., 1996 ). Stable transfectants expressing a similar level of chimeric protein were selected for use.

{blacksquare} MV binding assay.
The assay has been described previously (Gerlier et al., 1994 ). In brief, various amounts of purified MV Hallé were added to 2x105 cells in 50 µl DMEM supplemented with 6% foetal calf serum and 0·05% NaN3. After incubation at 37 °C for 60 min, the cells were washed and then incubated sequentially with an appropriate dilution of anti-H antibody and phycoerythrin-conjugated anti-Ig antibody. For analysis of MV binding, results were normalized for CD46 expression and expressed as a percentage of binding relative to wild-type CD46 (i.e. I–II–III–IV), which was set at 100%.

{blacksquare} Cell fusion assay.
The fusion capacity of each chimeric protein was determined by using a virus-based quantitative cell fusion-dependent reporter gene system (Alkhatib et al., 1996 ; Nussbaum et al., 1994 ). The first fusion partners (I–II–III–IV, x3DAF, x4DAF or x1/2DAF) were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (VVT7) (m.o.i. of 5). Simultaneously, the second fusion partner (I–II–III–IV) was infected with MV Hallé (m.o.i. of 5) and a recombinant vaccinia virus encoding the T7 promoter linked to the lacZ gene (VVlacZ) (m.o.i. of 5). After removal of non-adsorbed virus by washing, cells infected with MV were resuspended in culture medium containing 5 µg/ml of the fusion-inhibitory peptide zD–Phe–Phe–Gly (Richardson & Choppin, 1983 ). This ensured that any potential fusion between neighbouring cells expressing CD46 and the H and F proteins was inhibited. After overnight incubation at 37 °C and several washes at 37 °C to remove the fusion-inhibitory peptide completely, the cells were detached with a brief trypsin–EDTA treatment. The two fusion partners (2x105 cells in 200 µl containing 40 µg/ml cytosine arabinoside) were co-cultured at a ratio of 1:1 in a 96 well flat-bottomed plate. After incubation at 37 °C for 6 h, fusion was determined by the reporter gene-activation assay for {beta}-galactosidase with o-nitrophenyl {beta}-D-galactopyranoside as the colorimetric substrate. Results are expressed as a percentage of fusion relative to wild-type CD46 (i.e. I–II–III–IV infected with VVT7 plus I–II–III–IV infected with MV Hallé and VVlacZ), which was defined as 100%.

{blacksquare} Virus infectivity determination.
Cells were infected with MV Hallé (m.o.i. of 10) for 4 h at 37 °C. Non-adsorbed virus was removed and cells were incubated overnight at 37 °C in complete culture medium containing 5 µg/ml of the fusion-inhibitory peptide zD–Phe–Phe–Gly. One to four days post-infection, the percentage of cells expressing the MV H protein was determined by flow cytometry as described above.

{blacksquare} Down-regulation of CD46.
Cells were infected for 6 h at 37 °C with MV Hallé (m.o.i. of 1). Non-adsorbed virus was removed and cells were incubated overnight at 37 °C in the presence of zD–Phe–Phe–Gly as indicated above. Two days after infection, CD46 expression was determined by flow cytometry with the anti-CD46 MAb MCI20.6 and results were expressed as the percentage reduction in CD46 expression after infection compared with that observed in the absence of infection (Naniche et al., 1993b ).

{blacksquare} Cell surface distribution of CD46.
The cell surface expression and localization of I–II–III–IV and chimeric CD46 were assayed by two-stage immunofluorescence labelling. In brief, cells grown on coverslips were infected and treated as described above (see CD46 down-regulation). After incubation with MAb MCI20.6 at 4 °C and washing, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After additional washing, CD46 staining was revealed with a rhodamine-coupled anti-mouse Ig antibody. Analysis was performed with a Zeiss fluorescence microscope with a 40x objective.


   Results and Discussion
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Abstract
Introduction
Methods
Results and Discussion
References
 
CD46 SCR IV is required for optimal MV binding
To define further the accessory role(s) of the CD46 SCR III and IV domains with regard to MV binding, subsequent fusion, infectivity and CD46 down-regulation processes, chimeric proteins have been generated with the structurally related RCA protein DAF. The ability of the chimeric proteins to bind MV was determined by flow cytometry. Compared with wild-type CD46 (I–II–III–IV) (100% binding), a significant decrease in MV binding was observed with x4DAF ({approx}35% binding) (Fig. 1a), whereas a modest increase in binding was observed with x3DAF ({approx}120% binding). These findings were consistent over a range of MV dilutions. As a control, the binding of the x1/2DAF chimera was also determined. As expected, this protein, lacking CD46 SCRs I and II, did not have the ability to bind MV above the background binding observed on the parental CHO cells (<15% binding).



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Fig. 1. (a) MV binding to CD46-expressing CHO cells. CHO cells stably expressing wild-type CD46 (I–II–III–IV) or chimeric CD46 (x3DAF, x4DAF or x1/2DAF) were incubated with MV Hallé. Surface binding was detected by flow cytometry. Results are normalized for CD46 expression and expressed as a percentage of binding relative to I–II–III–IV, which was set at 100%. The data show the means (±SD) of three independent experiments. (b) Quantification of cell fusion observed following co-culture of CD46-expressing CHO cells (I–II–III–IV, x3DAF, x4DAF or x1/2DAF) infected with a recombinant vaccinia virus expressing T7 polymerase (VVT7) with cells (I–II–III–IV) infected with MV Hallé (m.o.i. of 5) and a recombinant VVlacZ. Results are expressed as a percentage of fusion relative to I–II–III–IV, which was set at 100%. The data show the means (±SD) of triplicate wells of two experiments. (c) Infection of CD46-expressing CHO cells (I–II–III–IV, x3DAF, x4DAF or x1/2DAF) with MV Hallé (m.o.i. of 10). Data are expressed as the percentage of cells infected by MV, which was determined by flow cytometry at 1 (open bars), 2 (hatched bars), 3 (cross-hatched bars) and 4 (filled bars) days post-infection by calculating the percentage of cells expressing the H protein. (d) CD46 down-regulation following infection with MV Hallé. CD46 expression was determined by flow cytometry approximately 48 h after MV infection (m.o.i. of 1) with the anti-CD46 MAb MCI20.6. Data are expressed as the percentage reduction in CD46 expression, calculated by comparing the levels of CD46 expression before and after infection. A representative experiment of three is shown.

 
The presence of SCR domains III and IV of CD46 has been shown previously to enhance MV binding (Devaux et al., 1997 ). The data presented above suggest that CD46 SCR IV is the dominant domain that is required for optimal virus binding, as isolated substitution of this SCR resulted in a significant reduction in MV binding. Unlike the CD46 SCR IV domain, the DAF SCR 4 domain is devoid of an N-linked oligosaccharide, and this could perhaps affect MV binding. MV binding to a carbohydrate-deficient CD46 mutant, obtained by mutating the N-glycosylation site in SCR IV (NST to QST) (Maisner et al., 1996 ; kindly provided by J. Atkinson, Washington University School of Medicine, St Louis, MO, USA), was in fact significantly increased ({approx}200%) (data not shown), indicating not only that the N-glycan on CD46 SCR IV is not required, but also that it appears to interfere somehow with MV binding.

Exchange of CD46 SCR III or IV does not effect fusion significantly
When MV-infected I–II–III–IV cells were used as the first fusion partner, a modest decrease in fusion relative to I–II–III–IV was observed with both x3DAF and x4DAF ({approx}75% of I–II–III–IV) (Fig. 1b). Thus, despite having a markedly reduced ability to bind MV (Fig. 1a), x4DAF was able to support efficient fusion (Fig. 1b). As a control, minimal fusion was observed with x1/2DAF (5%).

Both chimeric proteins allow MV entry
The ability of the chimeric proteins to allow MV infection was determined by flow cytometry. Two days after infection, {approx}35% of I–II–III–IV and x3DAF cells were infected; this was reduced to {approx}5% for x4DAF (Fig. 1c). The maximum number of x4DAF cells infected by MV was observed 3 days post-infection and represented {approx}15% of the cells (Fig. 1c). With the exception of I–II–III–IV, the percentage of cells infected was similar between days 3 and 4 p.i. Thus, both of the chimeric cells could allow MV entry. However, the percentage of infected cells was significantly lower with x4DAF, although the mean level of expression of H per infected cell was comparable to that observed with I–II–III–IV (data not shown). As a control, the infectivity of x1/2DAF was also determined. The maximum percentage of cells expressing H protein was within the background labelling of uninfected cells.

CD46 SCR IV plays an important role in MV-induced down-regulation
Besides directing MV binding and H+F-induced fusion, the MV–CD46 interaction also results in the down-regulation of cell surface CD46 (Naniche et al., 1993b ). MV infection resulted in a significant reduction in protein expression of I–II–III–IV ({approx}42%) and, to a lesser extent, x3DAF ({approx}22%). In contrast, no down-regulation was observed with x4DAF (Fig. 1d). This surprising observation was not restricted to a single x4DAF cell clone, as two other clones behaved in an identical manner, and similar data were also obtained when CD46 expression was determined using other anti-CD46 MAbs. Similarly, no down-regulation of the chimeric protein x1/2DAF was observed, since these cells expressed a protein devoid of both CD46 SCRs I and II, which have been demonstrated to be crucial for both MV binding (Buchholz et al., 1997 ; Iwata et al., 1995 ; Manchester et al., 1995 ) and CD46 down-regulation (Firsching et al., 1999 ). One simple explanation for the apparent lack of down-regulation of x4DAF could have been the lower level of expression of both the H and F proteins following MV infection ({approx}2–5-fold less) when compared with MV infection of I–II–III–IV and x3DAF (Fig. 1c and data not shown). However, and as observed with the x1/2DAF protein, the x4DAF protein was not down-regulated when a high level of H expression was achieved by infecting with a recombinant vaccinia virus encoding both MV H and F glycoproteins or H alone (data not shown). As a control, it was verified that no down-regulation of the I–II–III–IV protein was observed following infection with canine distemper virus (CDV), which, like MV, belongs to the genus Morbillivirus (data not shown). To exclude the possibility that the x4DAF and x1/2DAF proteins had lost their intrinsic ability to be down-regulated, anti-CD46 MAb MCI20.6 and anti-IgG antibody were used as alternative ligands to cross-link the hybrid CD46/DAF proteins. This induced a similar level of reduction in receptor expression ({approx}80%) with all of the CD46 chimeric proteins, thus excluding this hypothesis (data not shown).

Rearrangement of CD46 on the cell surface following MV infection
Strong CD46 staining was typically observed around the cell perimeter of non-infected cells, with a more diffuse punctuate pattern over the cell surface. This staining pattern was characteristic for I–II–III–IV and both of the chimeric proteins x3DAF and x4DAF (Fig. 2a, c). After MV infection of I–II–III–IV, the typical reduction in CD46 expression resulting from down-regulation was observed. However, the distribution of the remaining CD46 was clearly different, with a large proportion now being localized into discrete, but apparently random, patches on the cell surface (Fig. 2b). An almost identical staining pattern was observed with x3DAF following MV infection (data not shown). This pattern of staining is similar to the endocytosed intracellular patches of CD46 previously observed following MV H-induced down-regulation of CD46 (Naniche et al., 1993b ). In contrast, neither down-regulation nor patching of the receptor were observed following MV infection with x4DAF; the distribution pattern in these cells remained diffuse (Fig. 2d). Identical results were observed following infection with a recombinant vaccinia virus encoding both MV H and F glycoproteins, thus ruling out the possibility that this lack of rearrangement was due to the lower levels of these proteins (data not shown). Similarly, no ‘patching’ was observed with I–II–III–IV cells following CDV infection (data not shown).



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Fig. 2. Cell surface staining of CD46. Immunofluorescence analysis of CHO cells that stably expressed CD46 I–II–III–IV (a, b) or x4DAF (c, d), either non-infected (a, c) or after MV Hallé infection (m.o.i. of 1) (b, d). Analysis was performed with a Zeiss fluorescence microscope with a 40x objective.

 
As reported here, substitution of the CD46 SCR IV domain with the SCR 4 domain from the structurally and functionally related DAF protein resulted in a chimeric receptor with reduced MV binding and infectivity, and an insensitivity to both MV-induced down-regulation and cell surface redistribution. One could imagine that CD46 SCR IV exerts an effect indirectly by favourably modifying or maintaining an optimal conformation of the primary MV-binding site on SCRs I and II. Alternatively, the existence of a secondary MV-binding site on SCR IV cannot be excluded. An attractive hypothesis is that the SCR IV domain contains an oligomerization site that enables CD46 to oligomerize in response to the binding of the H tetramer protein (Malvoisin & Wild, 1993 ), thus strengthening MV binding and allowing the subsequent cell surface redistribution and down-regulation of CD46. This is reminiscent of the cellular receptor CD4 for human immunodeficiency virus, with a gp120-binding site localized in the N-terminal Ig-like domain and an oligomerization site localized in the membrane-proximal third and/or fourth domain(s) (Sakihama et al., 1995 ). It is interesting to note that the fourth Ig-like domain of CD4 was indeed present in all of the chimeric CD46–CD4 proteins where CD46 SCRs I and II were shown to be required for optimal contact-mediated down-regulation (Firsching et al., 1999 ). Perhaps this Ig-like domain can play a role that is functionally equivalent to the SCR IV domain of CD46, thus allowing oligomerization and subsequent down-regulation. This hypothesis of oligomerization through the SCR IV domain of CD46 is currently under investigation.

In conclusion, chimeric CD46/DAF proteins were constructed to investigate the accessory role(s) of SCR domains III and IV of CD46. The exchange of CD46 SCR IV (x4DAF) appears to have a marked effect on both MV binding and CD46 down-regulation. However, despite these observations, this protein was able to support fusion and allow MV infection. These data not only demonstrate the complexity that exists between the receptor and ligand, but also emphasize that the context of this interaction is critical. They also highlight the fact that one must exercise care when classifying a virus as being unable to use CD46 as a receptor on the basis of its inability to down-regulate CD46, as we have demonstrated clearly that down-regulation can be uncoupled from virus binding and entry.


   Acknowledgments
 
The authors thank all the members of Immunité et Infections Virales team for helpful discussions and D. Shafren and J. Atkinson for providing us with reagents. vCB21R-lacZ was obtained from Drs C. C. Broder, P. E. Kennedy and E. A. Berger through the AIDS Research and Reference Reagent program, Division of AIDS, NIAID. This work was supported in part by grants from CNRS, FNIMP and ARC (D.G.) and the NHMRC (Australia) (B.L., P.K., M.L.). D.C. is supported by a Marie Curie EU Fellowship. A French Government Scientific Fellowship during the initial stages of this work (D.C.) is also acknowledged.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
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Received 11 October 1999; accepted 6 January 2000.