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1 Institut für Virologie und Immunbiologie, Versbacher Straße 7, D-97078 Würzburg, Germany
2 School of Biomedical Sciences, The Queen's University of Belfast, Belfast BT9 7BL, UK
Correspondence
Jürgen Schneider-Schaulies
jss{at}vim.uni-wuerzburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Summers & Appel, 1994; Vandevelde & Zurbriggen, 1995). Recently, CD150 (SLAM) has been described as a cellular receptor for wild-type CDV (Tatsuo & Yanagi, 2002; Seki et al., 2003) and recombinant wild-type CDV has a similar tropism in ferrets as does Measles virus (MV) in humans (von Messling et al., 2004). However, as CDV readily infects epithelial cells and cells in the central nervous system, which are SLAM-negative (McQuaid & Cosby, 2002), it is likely that wild-type CDV uses additional receptors. Syncytium-forming and non-syncytium-forming strains of CDV have been isolated. The capacity to form syncytia in tissue culture appears to correlate with the attenuation of the viruses in natural hosts (Zurbriggen et al., 1995; von Messling et al., 2003). Infection and syncytium formation by the attenuated vaccine strain Onderstepoort in tissue culture do not depend on the expression of SLAM on target cells and, although SLAM improves its infectivity (Tatsuo et al., 2001), it is known that this strain and other tissue culture-adapted strains of CDV utilize additional, as-yet-unidentified receptors (Löffler et al., 1997).
We have shown that a mAb directed against CD9 (mAb K41) inhibits CDV-induced cell–cell fusion, but not virus–cell fusion (Schmid et al., 2000). However, direct binding of CDV to CD9 could not be demonstrated, suggesting that CD9 is not a receptor for CDV and that the effect on virus-induced cell–cell fusion is mediated via other cellular molecules. CD9 is a member of the tetraspanin transmembrane-protein (TM4) superfamily, which includes CD37, CD53, CD63, CD81, CD82 and CD151. These proteins form microdomains with a variety of other cell-surface receptors in the cell membrane (Hemler, 2003). CD9 has also been discussed as a possible cellular receptor for Feline immunodeficiency virus (FIV) (Willett et al., 1994). Similar to CDV, the infection of cells with FIV is inhibited by antibodies to CD9 in a step occurring after virus uptake (de Parseval et al., 1997; Willett et al., 1997). In contrast to our findings with CDV, these authors suggested that the release of FIV is affected by anti-CD9 antibodies. Another member of the TM4 superfamily, CD82, was identified to be involved in syncytium formation by human T-cell leukemia virus type 1 (HTLV-1) (Imai et al., 1992). Similar to our findings for CDV, neither a direct binding of FIV to feline CD9 nor of HTLV-1 to CD82 was demonstrated, again suggesting indirect functions of these two members of the TM4 family in virus release and cell-to-cell spread.
The efficiency of morbillivirus-induced cell fusion and virus release is influenced by the viral envelope proteins: the haemagglutinin (H), fusion (F) and matrix (M) proteins. Interacting with cellular receptors, the H protein determines the tropism and cytopathogenicity (von Messling et al., 2001). When cells are co-transfected with plasmids that express combinations of the H and F proteins of various CDV strains, the H protein determines the extent of cell–cell fusion. For MV, it has been demonstrated that mutations in the cytoplasmic domains of the H and F proteins (Cathomen et al., 1998a) and also the M protein influence the capacity of the virus to induce cell fusion, as a virus that does not express the M protein induced enhanced levels of cell–cell fusion (Cathomen et al., 1998b). In the light of these findings, we wanted to define the molecular basis of the anti-CD9 regulation of CDV-induced cell–cell fusion. Here, we describe that the CDV-H protein alone mediates the susceptibility to CD9 antibodies and that this effect is exerted by the extracellular domain of H.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Secondary antibodies goat anti-mouse and fluorescein isothiocyanate (FITC)- and horseradish peroxidase (HRP)-conjugated goat anti-rabbit were obtained from DAKO and Immunotech.
Cloning expression plasmids for viral envelope proteins.
The plasmids used for transfection of target cells were: pCG-CDV-H and pCG-CDV-F expressing the H and F proteins of CDV-OND-LP, pCG-MV-H and pCG-MV-F expressing the H and F proteins of MV-Edm, pCG-RPV-F expressing the F protein of the avirulent rinderpest virus (RPV) strain RBOK and pCG-F5804 expressing the F protein of the wild-type CDV strain 5804 (a gift of Dr V. von Messling, Montreal, Canada) (von Messling et al., 2001).
Restriction sites for XbaI and EcoRV were introduced at nucleotide positions 1686 and 2121, respectively, and the XbaI site at position 2691 was destroyed in pCG-CDV-H by using a site-directed mutagenesis kit (Invitrogen) by exchanging 1 or 2 nt according to the manufacturer's instructions. The nucleotide exchanges were chosen so that the amino acid sequences remained unchanged. The resulting plasmid was named pCG-CDV-H-XE. An EcoRV site was introduced into the plasmid pCG-MV-H at position 2010 by using the same method and the resulting plasmid was named pCG-MV-H-XE. The plasmids pCG-CDV-H-XE and pCG-MV-H-XE were taken to exchange the domains using the single restriction sites for BamHI, XbaI, EcoRV and SphI as described in Results (Fig. 3). The correct sequences of all clones were confirmed by sequencing using an ABI Prism 310 genetic analyser (Perkin-Elmer).
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Flow cytometry.
Flow-cytometric analyses were performed as described previously (Schneider-Schaulies et al., 1995). Briefly, cells (1x105) were incubated for 1 h on ice with 1 µg mAb in 100 µl FACS buffer (PBS containing 0.4 % BSA and 0.02 % sodium azide). Cells were washed twice in FACS buffer and incubated with 200 µl of a 1 : 100 dilution of FITC-conjugated goat anti-mouse immunoglobulin on ice for a further 1 h. After three washes with FACS buffer, flow-cytometric analysis was performed on a FACScan (Becton Dickinson).
Quantification of cell–cell fusion.
Vero cells (3x105) in a six-well plate were transfected with H- and F-expressing plasmids (1 µg each) with Lipofectamine 2000 (Invitrogen) and incubated for 24 h (or as indicated) at 37 °C. In the case of addition of mAb K41, it was added to the medium at indicated concentrations after transfection. Phase-contrast photomicrographs were taken of random regions by using a x20 objective with a digital camera (Leica). The number of nuclei in syncytia was counted and the mean number of nuclei in syncytia per well was calculated from several experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). As described previously (Löffler et al., 1997; Schmid et al., 2000), antibodies to CD9 (mAb K41) effectively inhibit the syncytium formation induced by infection of cells with CDV-OND (Fig. 1b). In contrast, syncytium formation induced by MV-Edm is not affected by CD9 antibodies (Fig. 1d).
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–h). Transfection of cells induced large syncytia, which were reduced in size in the presence of mAb K41. The level of inhibition with increasing concentrations of K41 was quantified based on the mean number of nuclei per syncytium (Fig. 1l). Transfection of cells with plasmids expressing MV-H and MV-F also induced the formation of very large syncytia. However, in contrast to the CDV envelope proteins, syncytium formation was not inhibited by mAb K41 (Fig. 1j, k; Table 1). These data demonstrate that CD9 antibodies specifically affect the functionality of the CDV envelope proteins H and F in the fusion assay and that the M protein and other viral proteins are not required.
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). The combinations of CDV-H with MV-F, RPV-F and wild-type CDV-F (CDV5804-F) were inhibited less effectively (46–67 %) than the CDV-H/CDV-F combination (83 %), whereas the combinations containing MV-H were not inhibited at all. Mean numbers of nuclei and percentages of inhibition by mAb K41 are shown in Table 1. These results demonstrate that, predominantly, the CDV-H protein mediates the susceptibility to the anti-CD9 antibody. In contrast, CDV-F can be substituted by F proteins of other morbilliviruses and the effect is still observed, although modulated by the various F proteins.
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), and whether it is mediated via signals from within the cell or whether the ectodomain of the H protein important for the dimerization (Plemper et al., 2000) and interacting with cellular receptors on the surface of cells (Langedijk et al., 1997; Massé et al., 2004) may play a decisive role, we generated chimaeric H proteins by exchanging parts of CDV-H and MV-H. To facilitate these exchanges, we introduced and ablated restriction sites. Nucleotide exchanges were chosen so that they did not change the amino acid sequences of the CDV-H and MV-H proteins. Three component domains of the H proteins, namely (i) the cytoplasmic plus transmembrane domain (residues 1–58), (ii) the stem structure (stem 1+barrel strand 1+stem 2; residues 59–227) and (iii) the remaining residues in the globular head domains of the CDV-H and MV-H proteins were exchanged (Langedijk et al., 1997). XbaI and EcoRV restriction sites were introduced as described in Methods. The four restriction sites BamHI, XbaI, EcoRV and SphI were used to exchange the domains (Fig. 3a). Expression plasmids with mutated restriction sites, pCG-CDV-H-XE and pCG-MV-H-XE, and those encoding chimaeric H proteins with exchanged domains were generated and designated pCG-CH-1 to -CH-6 as indicated (Fig. 3a). The sequences of all plasmids were confirmed by sequencing.
Total expression levels of the chimaeras were examined by Western blotting using polyclonal sera raised against 15 aa of the cytoplasmic tails of MV-H or CDV-H. The MV-specific serum was used to detect CH-1, CH-5, CH-6 and MV-H and the CDV-specific serum was used to detect CH-2, CH-3, CH-4 and CDV-H. The findings demonstrate that all chimaeric H proteins were expressed at similar levels (Fig. 3b). The molecular masses vary depending on the number of N-glycosylation sites and the slightly longer globular head domain of MV-H. As shown in Fig. 3(a), MV-H has four N-glycosylation sites in the XbaI–EcoRV fragment at amino acid positions 168, 187, 200 and 215 (Hu et al., 1994), whereas CDV-H has one site in this domain and one in the globular head domain, at amino acid positions 149 and 422 (Iwatsuki et al., 2000).
The presence of the H proteins on the cell surface was assessed by flow cytometry using mAbs to CDV-H (9E2) or MV-H (L77). We have previously mapped the mAb-binding site of L77 to positions 377–378 by escape mutations (Liebert et al., 1994; Moeller et al., 2001). The recombinant proteins CDV-H-XE and MV-H-XE (Fig. 3c) were expressed on the surface of transfected cells at levels similar to those of the transiently transfected parental CDV-H (OND-LP) and MV-H (Edm) proteins (not shown) and the chimaeric constructs (Fig. 3c). The finding that mAb 9E2 to CDV-H binds well to CH-2 indicates that its epitope, like that of mAb L77, is located in the globular head. The epitopes recognized by the mAbs are conformational and were also detected in the chimaeric molecules. This suggests that the conformation of the globular head of the chimaeric proteins is similar to those of the parental CDV-H and MV-H proteins and that the domain switches do not affect the recognized structure adversely.
We then tested the ability of the mutated and chimaeric haemagglutinins to induce syncytium formation and to be inhibited by mAb K41. After transfection of cells with plasmids encoding CDV-H-XE, MV-H-XE, CH-1 and CH-4 in combination with CDV-F, syncytia were formed (Fig. 4). In contrast, transfection of plasmids encoding CH-2, CH-3, CH-5 and CH-6, the chimaeras with heterologous ectodomains, did not lead to syncytium formation (not shown), which indicates that these heterologous extracellular domains are non-functional. When we tested the effect of the anti-CD9 mAb, we found that the syncytium formation induced by CDV-H-XE and CH-1 containing the ectodomain of CDV was inhibited, whereas the syncytium formation induced by CH-4 containing the transmembrane and cytoplasmic domain of CDV and the ectodomain of MV was not inhibited. The syncytia formed by this recombinant were smaller and developed more slowly than those induced by the parental molecules. However, there was clearly no inhibitory effect by the anti-CD9 antibody (Fig. 4e, f). Numbers of nuclei in syncytia and percentages of inhibition by mAb K41 are given in Table 2. Similar results were obtained when the parental and chimaeric H proteins were co-transfected with MV-F instead of CDV-F (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schmid et al., 2000). The antibody-induced inhibition of cell–cell fusion is virus-specific, as only CDV-induced cell fusion, but not cell fusion induced by other viruses such as MV (Fig. 1) or Human immunodeficiency virus 1 (not shown), is affected. This suggested that one of the viral envelope-associated proteins of CDV involved in membrane fusion may interact specifically with an anti-CD9-regulated molecule. The anti-CD9-mediated regulation of cell–cell fusion might occur via signal transduction or via a direct molecular interaction with membrane-associated host-cell molecules. However, as the antibody concentrations necessary for inhibition of fusion are relatively high (in the range of 5–15 µg ml–1), it is more likely that direct interactions are involved. There are several possibilities for how the anti-CD9 regulation may influence fusogenicity: the cellular molecule on which CD9 acts may influence cell fusion as a receptor for H, as enhancer/inhibitor of the F protein-mediated membrane fusion or as enhancer/inhibitor of the H–F or the H–F–M protein interaction. To come a step closer to answering the question of by which mechanism the inhibition of cell fusion may occur, we investigated which viral protein(s) is/are involved. By using transient-transfection assays, we have shown that the CDV-H protein determines the susceptibility of cell–cell fusion to anti-CD9 antibodies. The effect is modulated by the associated F protein, which may reflect variations in the strength or efficiency of the interaction between the CDV-H and F proteins of various origins.
The predicted structures of the MV-H and the CDV-H proteins are similar (Langedijk et al., 1997). It was therefore possible to exchange regions of the H proteins and to generate chimaeric molecules in order to determine which part of the molecule determined the inhibitory effect of the anti-CD9 antibody for CDV. Although all chimaeric H proteins were expressed at similar levels on the surface of cells, not all combinations supported cell–cell fusion. Non-functional chimaeras were CH-2, CH-3, CH-5 and CH-6, all of which are chimaeras with the stem structure and the globular head domain coming from different viruses. Thus, these combinations appear to lead to non-functional conformations either not recognizing the cellular receptors or lacking the proper conformation for the H–F interaction required for successful fusion (Wild et al., 1991). In contrast, chimaeras in which complete ectodomains are coupled to the combined cytoplasmic and transmembrane domains of the other virus are functional and induce cell–cell fusion in the presence of the F proteins of both CDV and MV. The cell–cell fusion induced by CH-1, containing the ectodomain of CDV, was inhibited by antibodies to CD9, whereas that induced by CH-4, containing the ectodomain of MV, was not. Thus, the extracellular domains of the H protein of CDV are necessary and sufficient to mediate the anti-CD9 modulation of cell–cell fusion. Our data suggest that the interaction of H with an unknown cellular receptor is affected and that CD9 antibodies regulate the activity and/or the spatial expression pattern of this receptor. This may occur by transport of the putative receptor to areas of the membrane that support virus–cell fusion (e.g. the upper surface of the cell, exposed to the medium), but not cell–cell fusion (e.g. the cell–cell contact areas), which would render the receptors inaccessable to the H proteins expressed on the cell surface of neighbouring cells.
Tetraspanin-family molecules are cell type-specifically organized in membrane microdomains with several other receptors, such as integrins and heparin-binding EGF-like growth factor (Hemler, 2003). CD9 plays a role in cell-fusion events in the absence of viruses and is involved in organogenesis (Hemler, 2003). It modulates the fusion of blood monocytes during the genesis of osteoclasts (Tanio et al., 1999), is involved in the fusion of myoblasts (Tachibana & Hemler, 1999) and regulates the fusion of gametes (Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000). The mechanism of CD9-induced regulation of receptor activity or distribution may also play an important role in these cellular processes. Further work is required to identify the receptor and the associated mechanism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 24 October 2005;
accepted 13 February 2006.
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