Interactions of decay-accelerating factor (DAF) with haemagglutinating human enteroviruses: utilizing variation in primate DAF to map virus binding sites

Abstract

A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 1113, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR₃₄ of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. & Lea, S. (2003). J Biol Chem 278, 1069110696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virusreceptor contacts.

We thank Dr Darren Shafren (University of Newcastle, Australia) for RD-ICAM1 cells. MAb 1H4 was kindly provided by Dr D. Lublin (Washington University School of Medicine, USA). This work was supported by a Medical Research Council programme grant (#G9901250) to D. J. E. and a project grant from The Wellcome Trust (#059011) to D. J. E. and S. L.

Footnotes

^,†, Ian G. Goodfellow¹ †Present address: School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, UK.

Many human enteroviruses, including representatives within the species human enterovirus B, C and D (HEV-B, HEV-C, HEV-D), bind decay accelerating factor (DAF; CD55), a 70 kDa member of the regulator of complement activity protein family (RCA) (Bergelson et al., 1994; 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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Fig. 1. (a) Schematic diagram of human DAF showing each of the short consensus repeat (SCR) protein domains and their functional activities. -N and -O respectively indicate N-linked or O-linked carbohydrates; GPI indicates the glycosylphosphatidylinositol anchor. (b) Alignment of human, orang-utan, African Green monkey (AGM) and baboon DAF SCR₁₂₃₄ and S/T-rich domain protein sequences. Residues that differ in non-human primate DAF are indicated, with identity shown with a period (.) and hyphens (-) used to indicate gaps introduced during alignment. Cysteine residues involved in the disulphide bonds characteristic of SCR domains are indicated in bold and asterisked, and the amino acid numbering of the mature protein is indicated in parentheses at the line ends. Sequences were aligned using ClustalW (Thompson et al., 1994) in BioEdit v5.0.9 (Hall, 1999). GenBank accession numbers for each of the sequences used are as follows: human DAF, P08174; orang-utan DAF, AAC60609; African Green monkey DAF, AA037580; baboon DAF, AAF73178.

We have investigated the interaction of a range of DAF-binding enteroviruses (CV-A21 and EV6#591, 7, 11-207, 12, 13 and 29) with primate cell lines from humans (RD), African Green monkeys (AGM; COS7), baboons (26CB1) or orang-utans (EB185), obtained from ATCC or ECACC. With the exception of CV-A21, which was propagated in RD cells stably expressing ICAM-1 (RD-ICAM1), all viruses were grown in RD cells. EV6#591 and EV11-207 are clinical isolates of EV6 and EV11 respectively and have been described previously (Goodfellow et al., 2001; 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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Fig. 2. (a) Binding of ³⁵S-labelled enteroviruses to primate cells with (pale bars) or without (dark bars) pre-incubation with anti-human DAF polyclonal antibody (pAb DAF). All viruses were partially purified following metabolic labelling by pelleting through a 30 % sucrose cushion essentially as previously described (Spiller et al., 2002). With the exception of EV13 and CV-A21, 160S virus particles were further purified by centrifugation through a 1025 % sucrose gradient (200 000 g in a Sorvall TH-641 rotor at 4 °C for 75 min). Following fractionation and scintillation counting, fractions corresponding to the 160S particles were again centrifuged (100 000 g in a Beckman TLS-55 rotor at 4 °C for 6 h) to remove sucrose and resuspended in PBS containing 0·1 % BSA. Previous studies have shown that there are no differences in the binding of partially purified (30 % sucrose cushion) EV7, 11-207, 12, 29 and 160S particles of the same viruses (D. T. Williams, unpublished data). Binding assays were performed as previously described (Goodfellow et al., 2001) using 10⁵ c.p.m. of 30 % cushion purified virus, or 2x10⁴ c.p.m. of 160S virus. Adherent cells (RD, CHO, COS7) were disaggregated with PBS/EDTA before use and 2·5x10⁶ cells were used in each assay. The results are presented as the mean of duplicate assays in percentage of radioactive virus bound (c.p.m.) relative to binding to untreated human RD cells. CHO cells were used as a negative control (Clarkson et al., 1995; Spiller et al., 2000). (b) Replication of DAF-binding enteroviruses in RD and COS7 cell lines. 1x10⁵ cells in 50 µl were mixed with an equal volume of the indicated virus (100 TCID₅₀) and allowed to bind for 1 h at 37 °C before the addition of 300 µl of serum-free media. After incubation for a further 3 days at 37 °C cells were freezethawed three times to release intracellular virus which was titrated in RD cells. Progeny EV11-207 and EV13 were not observed above the limit of detection (1 TCID₅₀) of the assay. RD-ICAM1 cells were substituted for RD cells for all assays in which CV-A21 was present. In parallel assays, set up in the same way, infected cell monolayers were fixed with ice-cold acetone/methanol at 8 h post-infection and stained using the generic anti-enterovirus mAb 5-D8/1 (Dako) and a secondary β-galactosidase-conjugated goat anti-mouse antibody (Harlan Sera-lab). Cells containing de novo synthesized enterovirus capsids stain blue following colour development with cyanide X-Gal solution [0·05 % X-Gal, 3 mM K₄Fe(CN)₆, 1 mM MgCl₂, 0·1 % BSA]. Infected cell monolayers staining blue are indicated as dark bars, those in which no new capsids were synthesized are shown as pale filled bars. COS7 cells infected with EV11207 or EV13 did not stain blue, and contained no infectious virus.

Current evidence indicates that DAF may act as an initial attachment receptor, with subsequent internalization requiring the presence of additional cell surface components or proteins (Powell et al., 1997, 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 β3 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 β4a strand and the loop that leads to the β4b 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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Fig. 3. (a) Alignment of the four SCR domains of human DAF indicating the residues contributing to the six beta strands characteristic of the SCR fold shown in over height text. Cysteine residues contributing to the SCR structure are highlighted in bold. The numbering of the mature protein is indicated in parentheses at the start of each domain. (b) Structure of SCR₃₄ of human DAF. Front and back views (rotated 180° about the vertical axis) of the protein are as recently defined (Williams et al., 2003). (I) Space-filled model of SCR1, based upon the known structure of SCR3 (Williams et al., 2003). The region highlighted in grey is missing from the available sequence for SCR₁ of orang-utan DAF, and include residues (in dark grey) that vary in other primate DAF proteins and are predicted to be important for binding CAV21 (see Fig. 1b). Arrows indicate the likely location of two insertions relative to the human sequence present in primate DAF SCR₁ that flank the β4a strand, which is highlighted in green. (II) SCR₃₄ structure indicating residues (in blue) implicated in the binding of EV6#591, EV11-207 and EV13 based upon the differences between human and primate DAF. Previous studies have demonstrated that Ser-165, Phe-169 and Val-215 (pale blue) are not involved in binding of these viruses (Williams et al., 2003), or are largely buried and so probably inaccessible to virus. (III) SCR₃₄ structure indicating residues (in dark blue) implicated in EV7 and EV29 binding based upon the differences between AGM and baboon DAF. The epitope for mAb 1H4 which blocks receptor binding by these viruses is highlighted in red (Hasan et al., 2002; unpublished results). (IV) SCR₃₄ structure showing residues that vary (dark blue) in alignments of primate DAF proteins (see Fig. 1b). The conserved front face of the protein is predicted to be the predominant site of EV12 interaction, a conclusion supported by the demonstration that mutation of Glu-134 (pale blue) reduces EV12 binding (Williams et al., 2003).

EV6#591, EV11-207 and EV13 only bind human DAF (Fig. 2a) 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 β3 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 β2 and β4b 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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Received 23 September 2003; accepted 4 November 2003.