1 Institut für Virologie und Immunbiologie, Versbacher Str. 7, 97078 Würzburg, Germany
2 Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
Correspondence
Jürgen Schneider-Schaulies
jss{at}vim.uni-wuerzburg.de
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ABSTRACT |
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INTRODUCTION |
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Following the identification of CD46 as a receptor for MV vaccine and laboratory strains (Dörig et al., 1993; Naniche et al., 1993a
), evidence has accumulated that many wild-type isolates do not use CD46 as a receptor. Recently, the signalling lymphocytic activation molecule (SLAM, CD150) has been identified as a common receptor interacting with MV vaccine as well as wild-type strains (Erlenhoefer et al., 2001
, 2002
; Hsu et al., 2001
; Ono et al., 2001a
, b
; Tatsuo et al., 2000
). SLAM is expressed on human B cell lines, primary activated B and T cells, memory cells and 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 a receptor can explain the tropism of wild-type MV for such cells, but not for epithelial, endothelial and neural cells, such as neurons, oligodendrocytes and astrocytes, which do not express SLAM (McQuaid & Cosby, 2002
). On monocytes, the expression of SLAM is induced after infection with MV or treatment with UV-inactivated MV (Minagawa et al., 2001
). Recently we demonstrated that this induction of SLAM is due to the interaction of wild-type MV with the Toll-like receptor 2, which is not a receptor for uptake of MV (Bieback et al., 2002
).
Several observations with epitheloid cell lines such as the African green monkey kidney cell line Vero suggest the presence of additional uptake mechanisms or unknown receptors on such cells (Hashimoto et al., 2002; Koumomou & Wild, 2002
; Nielsen et al., 2001
; Takeuchi et al., 2002
). We recently demonstrated that most wild-type MV and recombinant viruses expressing the envelope haemagglutinin (H) and fusion (F) proteins of these strains do not use CD46 as a receptor (Erlenhoefer et al., 2002
); however, they can infect SLAM-negative Vero and Hela cells (Johnston et al., 1999
), which supports the suggestion of CD46- and SLAM-independent virus uptake. Here we have demonstrated that primary human umbilical vein endothelial cells (HUVECs) and transformed human brain microvascular endothelial cells (HBMECs) do not express SLAM, either with and without treatment with inflammatory cytokines or MV. Since certain wild-type MV strains can use CD46 as a low-affinity receptor on the surface of lymphoid cells (Manchester et al., 2000
), we assessed whether CD46 might be involved in virus uptake by ECs. We found that, in the absence of SLAM and in the presence of CD46-blocking antibodies, wild-type MV could effectively infect ECs, suggesting the presence of an additional non-CD46/non-SLAM cellular receptor(s) for MV.
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METHODS |
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Virus strains and antibodies.
MVs used in this study were the vaccine-like strain Edmonston (Edm) and the wild-type strains WTFb, Wü4797 (Würzburg.DEU/96/4797) and Wü5679 (Würzburg.DEU/98/5679; Erlenhoefer et al., 2002). For further details see Table 1
. Edm was amplified using Vero cells and the wild-type strains using the human EpsteinBarr virus (EBV)-transformed B cell line BJAB, which does not produce EBV. Titres of all viruses were determined using the monkey EBV-transformed B cell line B95a (Kobune et al., 1990
).
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Immunohistochemistry and infection inhibition assay.
For immunohistochemistry, four- or eight-chamber glass slides (Nunc) were coated with 0·5 % gelatin and 2 % glutardialdehyde and washed three times with PBS prior to seeding of the cells. Cells were fixed for 7 min with 3·7 % paraformaldehyde and permeabilized for 10 min with 0·25 % Triton X-100. Non-specific antibody binding was blocked by incubation with 10 % FCS for 45 min at 4 °C. MV infection was detected using mAb F227 to MV N (2 µg ml-1) and goat anti-mouse IgG Alexa Fluor 488-conjugated secondary antibody (Molecular Probes). Cell nuclei were stained using DAPI nucleic acid stain (Molecular Probes).
For the infection inhibition assay, cells were incubated prior to infection with mAbs to CD46 at given concentrations at 37 °C for 1 h, infected at an m.o.i. of 0·1 with MV for 1 h, washed once with PBS and further incubated in culture medium supplemented with antibody for 2 days. MV was visualized using a human MV-specific hyperimmune serum of a SSPE patient and secondary FITC-conjugated swine anti-human antibodies.
RT-PCR.
Total cellular RNA was isolated using the GeneElute Mammalian Total RNA kit (Sigma). Reverse transcription was primed with oligo(dT) primers and carried out using SuperScript II RNase H-free reverse transcriptase (Gibco). The PCR primers were: SLAM, forward 5'-CTCCTCATTGGCTGATGGATCC-3', reverse 5'-TTTATGAGCAGGTCTCCACTCC-3'; CD46, forward 5'-TCGATACATATGGAGCCTCCCG-3', reverse 5'-CTAGGCCTACTTACAAGCCTCC-3'; -actin, forward 5'-TGACGGGGTCACCCACACTGTGCCC-3', reverse 5'-CTAGAAGCATTTGCGGTGGACGAT-3' and TLR2, forward 5'-GCCAAAGTCTTGATTGATTGG-3', reverse 5'-TTGAAGTTCTCCAGCTCCTG-3'. For the PCR, Ready To Go PCR beads (Amersham Pharmacia) were used.
Virus binding assay.
Cells (5x104) were incubated in 100 µl PBS at 37 °C for 1 h with viruses at a given m.o.i., washed with FACS buffer (Ca2+/Mg2+-free PBS containing 0·4 % BSA and 0·02 % sodium azide) and stained with mAb K83 against MV H and FITC-conjugated goat anti-mouse antibodies. Bound virus was determined by analysis with a FACScan (Becton Dickinson).
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RESULTS |
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Analysis of virus binding to and infection of HUVECs and HBMECs
As the first step of the infection cycle, MV binds to its cellular receptors. The amount of bound virus can be taken as an indicator of the presence of receptors on the cell surface. We therefore analysed the binding capacity of ECs for CD46-utilizing and non-utilizing MV strains. Viruses corresponding to m.o.i.s of 1, 2·5 and 5 (titrated using B95a cells) were incubated with HUVECs and HBMECs for 1 h and viral envelope proteins present on the cell surface were quantified by flow cytometry. The vaccine strain Edm, as expected, bound very efficiently to a high percentage of HUVECs and HBMECs because of its high affinity to CD46 (Fig. 2). In contrast, the wild-type strains WTFb, Wü4797 and Wü5679 bound to only a fraction of the cells. Interestingly, the efficiency of virus binding varied substantially between the wild-type strains. Reproducibly more HUVECs and HBMECs bound strain Wü4797 (up to 40 and 60 %, respectively) than the other two wild-type strains, WTFb and Wü5679 (up to 15 and 20 %, respectively).
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DISCUSSION |
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Monocytes/macrophages have previously been described as not expressing SLAM. However, it was recently found that activation of PBMCs with phytohaemagglutinin, LPS or MV particles leads to the induction of SLAM on CD14-positive cells (Minagawa et al., 2001). Interestingly, not only infection with MV but also UV-inactivated MV induced SLAM on such cells (Minagawa et al., 2001
). The basis for this observation was recently elucidated when we found that wild-type MV interacts with TLR2, an interaction that induces intracellular signalling via the transcription factor NF-
B and the expression of SLAM (Bieback et al., 2002
). While mediating the activation of monocytes by MV, TLR2 is not an entry receptor for the virus (Bieback et al., 2002
). Since ECs also express TLRs, which can be enhanced by LPS and IFN-
(Faure et al., 2001
), we asked whether treatment of the ECs with the TLR agonists LPS or MV could lead to the induction of SLAM. However, SLAM remained non-inducible on HUVECs and HBMECs.
In order to exclude a role for CD46 in the uptake of wild-type MV, we used strains that have been demonstrated to be unable to use CD46 as a receptor on the surface of transfected Chinese hamster ovary (CHO) cells (Erlenhoefer et al., 2002) and applied anti-CD46 antibodies to inhibit a potential interaction of wild-type MVs with this receptor on human ECs. The results indicated that MV wild-types can infect ECs in a CD46- and SLAM-independent manner. Receptor-independent mechanisms of virus spread, possibly as microfusion events at synapses, might circumvent the necessity for specific receptors for MV in the brain (Allen et al., 1996
; Duprex et al., 1999
; Lawrence et al., 2000
; McQuaid et al., 1998
; Meissner & Koschel, 1995
; Urbanska et al., 1997
), where CD46 is present on only a small proportion of cells and SLAM is not expressed (McQuaid & Cosby, 2002
; Ogata et al., 1997
). These findings are valid for virus spread in neurons in the brain, but not for infection of tissue culture cells with cell-free virus. The uptake of virus by ECs in tissue culture was relatively effective, which supports the assumption of a receptor-mediated process. Our data therefore suggest the presence of an additional unknown cellular receptor for MV on ECs. Our findings do not rule out the possibility that vaccine strains such as Edm may also use this additional receptor. We suggest this since, in the case of SLAM- and CD46-positive lymphocytes, virus attachment and infection with Edm can be blocked efficiently by antibodies to CD46, although Edm can also use the common MV receptor SLAM on cells in the absence of CD46 (Erlenhoefer et al., 2001
, 2002). The molecular basis for this finding is not known.
It currently remains unclear why certain wild-type strains spread better on HUVECs than others. Various evidence has been accumulated indicating that subtle differences in the envelope proteins of MV can play a role in altering the tropism, virus uptake, cell-to-cell fusion and pathogenicity (Bartz et al., 1996; Bieback et al., 2002
; Hsu et al., 1998
; Johnston et al., 1999
; Lecouturier et al., 1996
; Moeller et al., 2001
; Moll et al., 2001
; Ohgimoto et al., 2001
; Plemper et al., 2002
; Shibahara et al., 1994
; Takeuchi et al., 2002
). To investigate the molecular basis for the differential spread of MV wild-types in cultures of HUVECs, we will further analyse the sequences of the envelope genes of the MV wild-types and intend functional studies with corresponding recombinant viruses.
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ACKNOWLEDGEMENTS |
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Received 4 October 2002;
accepted 16 December 2002.