Analysis of receptor (CD46, CD150) usage by measles virus

Christian Erlenhöfer1, W. Paul Duprex2, Bert K. Rima2, Volker ter Meulen1 and Jürgen Schneider-Schaulies1

Institut für Virologie und Immunbiologie, Versbacher Str. 7, D-97078 Würzburg, Germany1
School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 7BL, UK2

Author for correspondence: Jürgen Schneider-Schaulies. Fax +49 931 2013934. e-mail jss{at}vim.uni-wuerzburg.de


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In order to investigate which measles virus (MV)-strains use CD46 and/or CD150 (signalling lymphocytic activation molecule, SLAM) as receptors, CHO cells expressing either recombinant CD46 or SLAM were infected with a panel of 28 MV-strains including vaccine strains, wild-type strains with various passage histories and recombinant viruses. We found that SLAM served as a common receptor conferring virus uptake and syncytium formation for all MV-strains tested. Predominantly vaccine and laboratory adapted strains, but also a minor fraction of the wild-type strains tested, could utilize both CD46 and SLAM. Using recombinant viruses, we demonstrate that the single amino acid exchange in the haemagglutinin (H) protein at position 481 Asn/Tyr (H481NY) determines whether the virus can utilize CD46. This amino acid alteration has no affect on the usage of SLAM as receptor, and as such demonstrates that the binding sites for SLAM and CD46 are distinct.


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Following the identification of complement regulatory molecule CD46 as a receptor for MV-strains Edmonston (Edm) and Hallé (Dörig et al., 1993 ; Naniche et al., 1993 ), evidence has accumulated that many MV-strains, especially wild-type isolates, do not use CD46 as a receptor (Buckland & Wild, 1997 ). Clear indication came from receptor modulation studies which revealed absence of CD46 downregulation after infection or contact with a number of wild-type MV-strains or recombinant MV expressing wild-type H (Bartz et al., 1996 ; Johnston et al., 1999 ; Lecouturier et al., 1996 ; Schneider-Schaulies et al., 1995a , b ). It has been found that some recent isolates of MV do not bind to or form syncytia in CD46-transfected cells, and that B cell lines such as BJAB or B95a express a different receptor for such viruses (Bartz et al., 1998 ; Hsu et al., 1998 ; Tanaka et al., 1998 ; Xie et al., 1999 ). The signalling lymphocytic activation molecule (SLAM, CD150) has been identified as this receptor, interacting with the MV vaccine strains Edm and Edm-Zagreb, and the wild-type strains KA, Montefiore89, WTFb and Wü5679 (Erlenhoefer et al., 2001 ; Hsu et al., 2001 ; Ono et al., 2001b ; Tatsuo et al., 2000 ). Other morbilliviruses such as canine distemper virus and rinderpest virus also use SLAM, but have a certain preference for their respective host SLAM homologues (Tatsuo et al., 2001 ).

Whether wild-type MV in vivo interacts with CD46 as a receptor or not, and whether all MV-strains can use SLAM as a receptor, is still not known. Addressing this question is important for a proper understanding of measles pathogenesis. One report described two clinical MV isolates, JW and IV, which interacted at low affinity with CD46. The infection of human PBMC with two further isolates, FV93 and BCL94, could not be inhibited with antibodies to CD46 (Manchester et al., 2000 ). The receptor usage of the latter strains could have been selected by using the SLAM-positive, CD46-negative cell line B95a for isolation. In contrast, another recent report supports the view that MV wild-types in throat swabs of acute measles patients all use SLAM and not CD46 (Ono et al., 2001a ). We therefore tested the receptor usage of as many as possible of the clinical isolates, strains and recombinant MVs available in our laboratory.

In order to determine the receptor usage we infected B95a cells (as positive control), parental CHO cells (as negative control), and CD46-positive and SLAM-positive CHO cells (Erlenhoefer et al., 2001 ). Testing CHO-CD46 and CHO-SLAM cells we can directly compare the effect of the receptors in the same cellular context. Cells infected for 2 days were fixed, permeabilized and stained with the monoclonal antibody F227 against the MV-nucleocapsid protein and secondary Alexa 488-conjugated antibodies (Molecular Probes) to analyse syncytium formation by immunofluorescence microscopy. At 2 days post-infection (p.i.) large syncytia were present in cultures of B95a and CHO-SLAM cells. SLAM-expressing cells generally formed syncytia faster than CD46-expressing cells, and therefore syncytia in CHO-SLAM and B95a cells are considerably larger than in CHO-CD46 cell at 2 days p.i. Results are shown in Fig. 1 and are summarized in Table 1. We observed only sporadically single infected cells in cultures in which the virus did not spread further (Fig. 1, CHO cells infected with BIL). In all cases in which specific receptors mediated virus entry, this was followed by the formation of (smaller or larger) syncytia.



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Fig. 1. Syncytium formation by B95a, CHO, CHO-CD46 and CHO-SLAM cells after infection with various MV strains and recombinants. Cells were infected with the indicated viruses for 2 days at an m.o.i. of 0·1, fixed with 3·7% paraformaldehyde, permeabilized with 0·2% Triton X-100, and stained with antibodies to the MV-nucleocapsid protein and Alexa 488-conjugated secondary antibodies. Magnification: 40x. Note that mostly large syncytia containing more than 20 nuclei are seen after infection of B95a and CHO-SLAM cells. Single infected cells are detected sporadically, as for example seen after infection of CHO cells with BIL.

 

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Table 1. Infection of B95a, CHO, CHO-CD46 and CHO-SLAM cells with MV-strains

 
All MV-strains, isolates or recombinants infected B95a cells and CHO-SLAM cells, and thus use SLAM as cellular receptor. In contrast, CD46 was used as receptor in addition to SLAM by a subset of the viruses including the vaccine strains Edm, Edm-Zagreb, Aik-C and genetically similar strains (Table 1, nos 1–6), and laboratory strains which all have been propagated using Vero cells (Table 1, nos 7–11). So-called wild-type isolates propagated using Vero cells, such as Chicago-1 (Chi-1), EBL, EBT and Kohno (Table 1, nos 12–15) also used both CD46 and SLAM. We did not find an MV using exclusively CD46 as receptor. Most wild-type viruses isolated using human B cell lines (BJAB, B-LCL), which express both CD46 and SLAM (Table 1, nos 16–22), exclusively used SLAM as receptor. An exception was the isolate BIL (Table 1, no. 16), which interacted with CD46 in addition to SLAM, and induced small syncytia on CHO-CD46 and larger syncytia on CHO-SLAM cells. The wild-type strain WTFb propagated using BJAB cells infected exclusively CHO-SLAM cells. After adaptation of the original WTF-isolate to growth in Vero cells (Rima et al., 1997 ) infection of cells with this strain (designated WTFv) was inhibited by antibodies against CD46, and both strains WTFb and WTFv did not induce the downregulation of CD46 from the surface of cells (Bartz et al., 1998 ). Here we show that WTFv (Table 1, no. 23) infected and formed syncytia on CHO-CD46 and CHO-SLAM cells (Fig. 1).

It is known that the amino acid at position 481 in MV-H has a strong influence on CD46 downregulation, CD46-dependent syncytium formation and haemagglutination (Bartz et al., 1996 ; Hsu et al., 1998 ; Lecouturier et al., 1996 ). All of these studies were performed using H-transfected cells and we therefore extended this study to examine the role of this residue for receptor interaction in the context of infectious virus. Thus, we tested recombinant MVs expressing defined envelope proteins and the remaining viral proteins of the Edmonston B strain. Edtag (no. 24) expresses F and H of strain Edm (Radecke et al., 1995 ), whereas MV(WTF-F/H)Ed expresses the F and H (no. 25), MV(WTF-F)Ed the F (no. 26) and MV(WTF-H)Ed the H (no. 27) of strain WTFb (Johnston et al., 1999 ). A point mutation was introduced in the WTFb H gene at nucleotide position 1441 (A to T), leading to amino acid Tyr instead of Asn at position 481 of the H protein of WTFb, to introduce the capacity to interact with CD46. A recombinant virus MV(WTF-H481N-Y)Ed (no. 28) containing this mutation was cloned and rescued according to a strategy described by Duprex et al. (1999a ) and was propagated on Vero cells. The growth characteristics of the parental virus Edtag and MV(WTF-H481N-Y)Ed were examined, and it was found that the new MV recombinant grows as well as Edtag in Vero cells (Fig. 2). This indicates that the interaction between the mutated wild-type H and the Edm F protein is functional and does not lead to any significant change in the growth kinetics of the virus.



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Fig. 2. Growth curves of the recombinant MVs Edtag and MV(WTF-H481N-Y)Ed. Vero cells were infected with the recombinants at an m.o.i. of 0·01. Cell-associated virus was prepared by freezing–thawing of the infected cells and titrated using B95a cells. Both viruses grow to similar titres.

 
The recombinant viruses (nos 24 and 26) expressing Edm H infected CHO-CD46 and CHO-SLAM cells. The wild-type F protein expressed in combination with the Edm H by MV(WTF-F)Ed was less fusogenic on CHO-CD46 cells in comparison to the H/F pairs in the viruses Edm and WTFv (Fig. 1). In contrast, the recombinants expressing the WTFb H (nos 25 and 27) infected only SLAM-positive cells. In addition, the recombinant MV(WTF-H481N-Y)Ed (no. 28) expressing the H protein of WTFb mutated at amino acid position 481 from Asn to Tyr, and thus restoring the capacity to interact with CD46, infected CHO-CD46 and CHO-SLAM cells. This demonstrates for the first time that a single point mutation in an infectious MV can change its receptor usage.

SLAM is expressed on human B cell lines, primary activated B and T cells, memory cells, activated monocytes and monocyte-derived dendritic cells (Cocks et al., 1995 ; Minagawa et al., 2001 ; Ohgimoto et al., 2001 ; Polacino et al., 1996 ; Punnonen et al., 1997 ), and its usage as receptor can explain the lymphotropism of wild-type MV. The data presented here and before (Erlenhoefer et al., 2001 ; Ono et al., 2001a ; Tatsuo et al., 2000 ) suggest that SLAM is a receptor for all MVs. The differential binding of certain MVs to CD46, especially when only the amino acid at position 481 is exchanged, does not influence the capacity of the viruses to interact with SLAM. This indicates that the SLAM binding site of MV-H is distinct from the CD46 binding site. According to the structural model of the H protein, amino acid 481 is located at the bottom of a small cleft in the fourth ‘propeller’ of the globular top of the H protein (Langedijk et al., 1997 ). Our data support the view that changes of this amino acid do not cause significant gross conformational changes.

The infection of epithelial, endothelial and neural cells by MV during acute and persistent infections cannot by explained on the basis of an exclusive usage of SLAM as receptor. Other mechanisms of virus spread might circumvent the need for specific receptors, especially in the brain, where SLAM cannot be detected in neurones, oligodendrocytes and astrocytes, and CD46 is expressed only by few cells (Ogata et al., 1997 ). A CD46-independent cell-to-cell spread of MV has been suggested to occur in the brain of SSPE (subacute sclerosing panencephalitis) patients, and has been found in corresponding animal models or tissue culture systems (Allen et al., 1996 ; Duprex et al., 1999b ; Lawrence et al., 2000 ; McQuaid et al., 1998 ; Meissner & Koschel, 1995 ; Urbanska et al., 1997 ). The infection of epithelial cells of the upper respiratory tract, and microvessels and underlying epithelial cells in the skin causing the rash could best be explained by the usage of CD46 as receptor, which, however, has not been found to be the case for most wild-type isolates and might require in vivo adaptation to CD46 receptor usage. The investigation of this question requires more experimental work.


   Acknowledgments
 
We thank Franziska Dimpfel and Sieglinde Löffler for technical assistance, and the Deutsche Forschungsgemeinschaft for financial support.


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Received 14 December 2001; accepted 4 February 2002.