Institut für Virologie und Immunbiologie, Versbacher Str. 7, D-97078 Würzburg, Germany1
School of Biology and Biochemistry, The Queens 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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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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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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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.
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References |
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Received 14 December 2001;
accepted 4 February 2002.