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Short Communication |
1 Faculty of Biomedical and Life Sciences, Division of Virology, University of Glasgow, Glasgow G11 5JR, UK
2 Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK
Correspondence
David J. Evans
David.Evans{at}vir.gla.ac.uk
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ABSTRACT |
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] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus–receptor contacts.
Present address: School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, UK. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Karnauchow et al., 1996; Lublin & Atkinson, 1989; Powell et al., 1999; Shafren et al., 1995; Ward et al., 1994). DAF has four short consensus repeat (SCR) domains, attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor and separated from it by a heavily O-glycosylated serine and threonine-rich domain (Fig. 1a). Structures of SCR34 and SCR23 have recently been published (Uhrinova et al., 2003; Williams et al., 2003), as has a cryo-EM complex of echovirus type 7 (EV7) with DAF (He et al., 2002). DAF-binding enteroviruses haemagglutinate erythrocytes and haemagglutination inhibition (HAI) using soluble DAF (sDAF) or SCR-specific monoclonal antibodies (mAb), together with virus binding or infection inhibition assays in the presence of sDAF or mAbs, have been used to map the SCR domain specificity of binding. With the exception of coxsackie A virus type 21 (CV-A21) and enterovirus type 70 (ENV70) which bind SCR1, all enteroviruses predominantly bind SCR3 with contributions from either or both of SCR2 and SCR4 (Clarkson et al., 1995; Karnauchow et al., 1998; Lea et al., 1998; Powell et al., 1999; Shafren et al., 1997a, 1998). The complement regulatory functions of DAF require SCR234 (Brodbeck et al., 1996; Coyne et al., 1992), whereas interaction with another known ligand, the leukocyte activation-induced antigen CD97, only involves SCR1 (Hamann et al., 1996, 1998). In the present study we utilize the inherent variation existing between primate DAF proteins (Kuttner-Kondo et al., 2000) to more precisely map regions of the receptor bound by DAF-binding human enteroviruses. This study complements our recent determination of the structure of SCR34 of human DAF in which limited mutagenesis was done to define functional regions of the protein (Williams et al., 2003).
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; Lea et al., 1998; Patel et al., 1985). African Green monkey (AGM) DAF was amplified by PCR and sequenced (GenBank accession no. AY178110), prior to alignment with other primate sequences (Fig. 1b
). Cell lines were all shown to express DAF by flow cytometric (FACS) analysis using rabbit anti-DAF polyclonal antisera (pAb DAF), and conservation of the epitopes for the domain specific mAbs MBC1 (SCR2), 1H4 or 854 (SCR3) was similarly tested. The epitope for mAb 854 was conserved on baboon, AGM and orang-utan DAF; mAb MBC1 failed to bind any non-human cell lines and mAb 1H4 did not bind AGM DAF on COS7 cells (data not shown).
Virus binding to primate cell lines was determined using 35S-metabolically labelled and purified virus stocks. Chinese hamster ovary (CHO) cells, known not to bind the viruses used in this study (Clarkson et al., 1995; Spiller et al., 2000), were used as a negative control. DAF-dependence was determined by pre-incubating the cells with pAb DAF and the results are presented in Fig. 2(a). EV6#591, EV11-207, EV13 and CV-A21 only exhibited significant levels of DAF-dependent binding to RD (human) cells, implying that these viruses interact with one or more regions unique to human DAF. EV12, a virus known to predominantly interact with SCR34 (Powell et al., 1999), bound to all the cell lines tested. EV7 and EV29 bound AGM (COS7) and human (RD) cells but did not bind baboon (26CB1) or orang-utan (EB185) lines, suggesting that these viruses interact with epitope(s) conserved between – and unique to – human and AGM DAF. The enhanced binding of some viruses to AGM cells reflects the higher levels of DAF that this line expresses, as determined by flow cytometry using cross-reactive pAb DAF (data not shown).
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, 1998). CV-A21 binds DAF but requires ICAM-1 for infection and the DAF-binding coxsackie B viruses (CV-B1, 3 or 5) need the coxsackie and adenovirus receptor (CAR) for entry (Shafren et al., 1997a, b; Shieh & Bergelson, 2002). To determine whether the cell-binding phenotypes of viruses measured above reflected the ability of the cell to support infection, a cell infection assay was conducted. Although normally cytopathic, enterovirus replication does not always result in cell lysis, so infection was monitored by virus yield, and in the case of adherent cell lines (RD and COS7) by a immunocolorimetric capsid detection assay (‘blue cell assay’; Spiller et al., 2002), which specifically stains cells in which de novo capsid synthesis has occurred (Fig. 2b and Spiller et al., 2002). All viruses replicated in RD cells (RD-ICAM1 cells were used for CV-A21) and caused a full cytopathic effect (cpe). Prior to the induction of a cpe, all infected RD cells could be stained blue using mAb 5-D8/1 (DAKO) in a ‘blue cell assay’, thereby demonstrating that this antibody could detect capsid proteins from these viruses. In contrast to these results, no cytopathology was apparent in the baboon (26CB1), orang-utan (EB185) and rodent (CHO) cell lines, and virus present at 72 hours post-infection was either undetectable or at a level commensurate with being carry-over from the initial inoculum (data not shown). Being suspension cell lines (which clump under the conditions required for immunocolorimetric screening) it was not possible to monitor de novo capsid synthesis in these lines. Although none of the viruses induced cpe in COS7 cells (AGM), virus was detectable at a level above or equal to the inoculum after infection with EV7, 12 and 29, but not with EV6, EV11-207, EV13 or CV-A21. Replication of EV7, 12 and 29 in COS7 cells was confirmed using the ‘blue cell assay’ (Fig. 2b). The absence of high levels of progeny EV7, 12 and 29 virus in the supernatant of COS7 cells, together with the intracellular staining of de novo synthesized capsids in this cell line, suggest that a late stage in the virus replication cycle such as encapsidation or particle egress may be inhibited in these infections. The viruses tested in this study can be divided into three distinct groups. EV6#591, EV11-207, EV13 and CV-A21 only bind DAF of human origin, and – of the lines tested – only replicate in RD cells. EV7 and EV29 form the second group, which bind to RD (human) and COS7 (AGM) cells in a DAF-dependent manner and also replicate in these cell lines. EV12 is the sole representative of the third group of viruses which displays DAF-dependent binding to all the primate cell lines used in this study, although it only infects and replicates in RD and COS7 cells. This suggests that there may be additional cell-surface or intracellular determinants lacking from the baboon and orang-utan cell lines that are required for infection.
The virus binding data, the natural variation in primate DAF proteins (Fig. 1b), the recently published structures of SCR34 and SCR23 (Uhrinova et al., 2003; Williams et al., 2003) and our site-directed mutagenesis of SCR34 (Williams et al., 2003) can together be used to map the regions of DAF involved in virus interaction more accurately than previously possible. SCR1 is the least conserved domain of DAF between the primate proteins used in this study. CV-A21 binds SCR1 of human DAF (Shafren et al., 1997a), but does not bind orang-utan DAF expressed on the EB185 cell line (Fig. 2a). There are three amino acid differences (Val-20, Met-29 and Lys-34; Fig. 1b) between human SCR1 and the published sequence for orang-utan DAF. By comparison of aligned SCR domains (Fig. 3a) Val-20 is located towards the beginning of the 
3 strand, but is not surface exposed and so is unlikely to contribute directly to binding. Orang-utan Met-29 and Lys-34 are respectively located in the short 4a strand and the loop that leads to the 4b strand. This region is located towards the base of the back face of SCR1, with Met29 situated close to the SCR12 interface. The first seven residues of orang-utan DAF have not been determined (Fig. 1b and Nickells et al., 1994). Both AGM and baboon DAF have differences in this region when compared to the human protein (Fig. 1b), which forms a poorly defined strand that delineates the front face of SCR1. Precise mapping of the binding site for CV-A21 will require the N-terminal sequence of mature orang-utan DAF and site-directed mutagenesis of residues that differ from human DAF. However, the location of these differences to the presumed membrane-proximal region of SCR1, or the side of this SCR domain, suggest that – if membrane-associated DAF is a linear protein – the virus is likely to interact in a side-on manner. This therefore differs from other picornaviruses that bind the N-terminal domains of other well characterized receptors such as in the poliovirus/PVR and rhinovirus/ICAM-1 interactions in which the membrane-distal portion of the receptor ‘docks' with a canyon on the virus surface. Alternatively, and possibly supported by the lack of any observed requirement for SCR234 by SCR1-binding enteroviruses, cell surface expressed DAF may not be linear, but instead may be kinked to present the side of SCR1 away from the membrane, in a conformation accessible to the virus.
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) and are known to require SCR234, but not SCR1, for binding (Lea et al., 1998; Powell et al., 1999). It is therefore likely that residues that differ between human DAF and DAF expressed on AGM, baboon and orang-utan cell lines may account for the differences in binding. These residues include Ser-72, Pro-107, Pro-143, Gly-144, Ser-165, Phe-169, Val-1215 and Asn-237 (Fig. 1b). In our recent analysis of DAF structure and function Ser-165 and Phe-169 were substituted for alanine without altering the ability of EV11-207 to bind to DAF (Williams et al., 2003). Similarly, sDAF bearing these mutations does not inhibit haemagglutination by EV6#591 or EV13 (data not shown). Of the remaining changes, Val-215 – situated in the 3 strand of SCR4 – is buried and is unlikely to influence virus binding. In contrast, Pro-143, Gly-144 and Asn-237 are at least partially exposed in the structure of SCR34 (Fig. 3b and Williams et al., 2003). Aligned SCR sequences (Fig. 3a) and the recent solution structure of SCR23 (Uhrinova et al., 2003) indicate that Ser-72 and Pro-107 are located at the amino termini of the 2 and 4b strands of SCR2 respectively. Although the lack of interdomain NOE constraints in the SCR23 structure do not allow the relative orientation of the SCR domains to be determined (Uhrinova et al., 2003), our preliminary crystal structure of SCR1234 indicates that Ser-72 is located on the same face of DAF as Pro-143 and Gly-144 in SCR3 and Asn-237 in SCR4 (Fig. 3b and data not shown). We predict that EV6#591, EV11-207 and EV13 interact with residues in SCR234 that define the right-hand face of the protein (using the orientation shown in Fig. 3b and defined in Williams et al., 2003). Site-directed mutagenesis of the individual residues highlighted above will be required to precisely define the virus contacts, and to determine whether these three viruses all interact in an identical manner with DAF.
EV7 and EV29 only bind RD (human) and COS7 (AGM) cells (Fig. 2a). Since these viruses do not bind baboon cells or require SCR1 for binding (Powell et al., 1999), one or more of the differences between human/AGM and baboon DAF within SCR234 must be implicated in virus binding. Of the six differences between SCR234 in these primate DAF protein sequences (Glu-65, Lys-116, Thr-151, Gly-174, Glu-205 and Asn-237), only three (Lys-116, Thr-151 and Gly-174) are conserved in human and AGM DAF, but differ in the baboon sequence (Fig. 1b). Two of these changes (Thr-151 and Gly-174) are located towards the membrane-distal end of SCR3, whilst Lys-116 is located towards the top of SCR2 (Uhrinova et al., 2003). A previous mutagenesis study has demonstrated that alanine substitution of Phe-169 – a residue located in close proximity to Thr-151 and Gly-174 – blocks the binding of mAb 1H4, an antibody known to inhibit infection by EV7 and 29 (D. T. Williams & Y. Chaudhry, unpublished results; Bergelson et al., 1994; Clarkson et al., 1995). Additional residues forming the mAb 1H4 epitope were recently mapped to Phe-148, Ser-155 and Leu-171 (Hasan et al., 2002). These results are consistent and suggest that a major region of interaction between DAF and both EV7 and 29 involves the top of the back face of SCR3 (Fig. 3b). Interestingly, the Phe-169 to alanine substitution enhanced the binding of EV7, but did not affect that of EV29 (Williams et al., 2003; and Y. Chaudhry, unpublished results), demonstrating that although these viruses are predicted to bind to the same region of DAF, subtle differences in these interactions exist. This is further supported by our previous observation of differences between EV7 and EV29 in their response to sDAF in HAI assays (Powell et al., 1999).
EV12 was distinct from the other viruses used in this study in that it bound all the primate cell lines tested. Surface plasmon resonance previously showed that interaction of this virus with human DAF is distinctly different from that of other echoviruses in that it predominantly involves SCR34 (Powell et al., 1999). Alanine replacement of Glu-134 reduced EV12-mediated haemagglutination, but not haemagglutination by EV7 or EV11-207 (Williams et al., 2003), nor the remaining viruses used in this study (data not shown). Glu-134 is conserved in primate DAF (Fig. 1b) and is located roughly centrally on the front face of SCR3, towards the top of a region that also encompasses the majority of SCR4, the face of which is highly conserved between the human and primate DAF proteins (Fig. 3b). We speculate that this sequence identity may reflect conservation of function in the interaction with the convertases. We interpret our results as indicating that EV12 binds to this conserved face of DAF – roughly defined as the front of SCR4 and the front of the membrane-distal half of SCR3 – a region distinctly different from the regions we predict as being important in binding the other viruses used in this study.
DAF binding is a widespread phenotype within the human enteroviruses. We demonstrate here that not only do certain viruses bind to different domains of the receptor, but that even viruses predominantly interacting with the same domain may interact with different faces of the protein. More accurate mapping of the interaction of virus and receptor will require a combination of cryo-EM, crystallographic analysis and site-directed mutagenesis. The observed differences in the virus binding site(s) on DAF shown here, the binding of DAF to the twofold axis of particle symmetry (He et al., 2002) and the inability of soluble DAF to trigger irreversible conformational changes in the virus particle (Powell et al., 1997), suggest that this interaction may have evolved to facilitate infection, perhaps by subversion of one of the cellular function(s) of DAF. Studies are under way to better understand the significance of this widespread interaction.
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ACKNOWLEDGEMENTS |
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Received 23 September 2003;
accepted 4 November 2003.
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