Unexpected Mode of Engagement Between Enterovirus 71 and Its Receptor SCARB2

LETTERS https://doi.org/10.1038/s41564-018-0319-z Unexpected mode of engagement between enterovirus 71 and its receptor SCARB2 Daming Zhou 1,7, Yuguang Zhao 1,7, Abhay Kotecha 1,2, Elizabeth E. Fry 1, James T. Kelly3,4, Xiangxi Wang5, Zihe Rao5, David J. Rowlands3, Jingshan Ren 1* and David I. Stuart 1,6* Enterovirus 71 (EV71) is a common cause of hand, foot critical to infectivity and can control virus tropism at both the spe- and mouth disease—a disease endemic especially in the cies and tissue level13. This makes receptor–virus interactions attrac- Asia-Pacific region1. Scavenger receptor class B member 2 tive targets for antiviral therapeutics, since it may be more difficult (SCARB2) is the major receptor of EV71, as well as several for a virus to acquire resistance to such a compound than to a classic other enteroviruses responsible for hand, foot and mouth dis- enzyme inhibitor. Recently, a number of receptors have been identi- ease, and plays a key role in cell entry2. The isolated structures fied for many of the aetiological agents of HFMD. Notable receptors of EV71 and SCARB2 are known3–6, but how they interact to include scavenger receptor class B member 2 (SCARB2; a receptor initiate infection is not. Here, we report the EV71–SCARB2 for EV71, CV-A16 and a subgroup of type A enteroviruses)2,14, krin- complex structure determined at 3.4 Å resolution using cryo- gle-containing transmembrane protein 1 (KREMEN1) for another electron microscopy. This reveals that SCARB2 binds EV71 on group of type A enteroviruses including CV-A10 (ref. 15), P-selectin the southern rim of the canyon, rather than across the can- glycoprotein ligand-1 for EV71, and coxsackievirus and adenovirus yon, as predicted3,7,8. Helices 152–163 (α5) and 183–193 (α7) receptor (CAR) and decay-accelerating factor (DAF) for group B of SCARB2 and the viral protein 1 (VP1) GH and VP2 EF loops enteroviruses16. Receptor usage correlates with the capsid struc- of EV71 dominate the interaction, suggesting an allosteric ture, indicating that receptor switching drives evolution (Fig. 1a). mechanism by which receptor binding might facilitate the In the absence of high-resolution structures for receptor–EV71 low-pH uncoating of the virus in the endosome/lysosome. complexes, it has been inferred that SCARB2 binds across the so- Remarkably, many residues within the binding footprint are called canyon—a depression that in enteroviruses encircles the not conserved across SCARB2-dependent enteroviruses; icosahedral fivefold axes and harbours the binding sites for slender however, a conserved proline and glycine seem to be key resi- immunoglobulin-domain-based receptors17, although SCARB2 is a dues. Thus, although the virus maintains antigenic variability much bulkier molecule. even within the receptor-binding footprint, the identification SCARB2, also known as lysosomal integral membrane pro- of binding ‘hot spots’ may facilitate the design of receptor tein-2, is a type III membrane protein with amino (N)- and car- mimic therapeutics less likely to quickly generate resistance. boxy (C)-terminal transmembrane helices18. It is found especially in Hand, foot and mouth disease (HFMD) infects mainly infants lysosomal limiting membranes and its 400-residue luminal domain and children, and has caused repeated epidemics in the Asia-Pacific is heavily glycosylated with nine potential N-linked glycosylation region for more than 20 years9, with around two million cases every sites6. SCARB2 has a major role in endo/lysosomal membrane year since 2010. While coxsackievirus A16 (CV-A16) and EV71 organization, with mutations causing several neurodegenerative are major aetiological agents of HFMD, a variety of viruses in the and renal diseases. There is good evidence that SCARB2 attach- genus Enterovirus, including many other type A and some type B ment mediates EV71 internalization and uncoating19,20. However, it enteroviruses, also cause the disease1. HFMD usually leads to rela- has been established that uncoating requires not only attachment to tively mild symptoms, such as fever, oral ulcerations and swellings SCARB2, but also low pH3. This implies that binding to SCARB2 on the hands and feet. However, EV71 infection is sometimes asso- might destabilize the virus particle at low pH (leading to the forma- ciated with cardiac and central nervous system complications and tion of expanded or ‘A-particles’5,11). Our aim was to visualize the even death1. Enteroviruses belong to the picornavirus family of initial attachment complex, so we used a variant of EV71 genotype icosahedral, unenveloped viruses10. They contain a positive-sense B2 whose infectivity is enhanced at low pH by a single mutation in single-stranded RNA genome, which on release into the cytoplasm VP1 (N104S; see Methods). The particles were further stabilized is directly translated by host ribosomes, initiating infection. Initial by replacing the natural pocket factor with a potent expansion stages of infection involve attachment to a host cell receptor, inter- inhibitor, NLD (see Methods). We determined the structure of the nalization, uncoating (which for enteroviruses is presumed to occur luminal domain of SCARB2 in complex with this stabilized form via expansion of the particle following ejection of a lipid pocket fac- of EV71 at a pH of 5.1 by cryo-electron microscopy (cryo-EM; see tor from the VP1 β -barrel resulting in a cascade of structural rear- Methods and Supplementary Fig. 1a). We found that SCARB2 binds rangements11) and release of the genome through a membrane pore to the ‘southern rim’ of the canyon, interacting with loops from two into the cytoplasm12. Correct engagement with a specific receptor is of the major capsid proteins, VP2 and VP1 (Fig. 1b). The structure 1Division of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. 2Materials and Structural Analysis, Thermo Fisher Scientific, Eindhoven, the Netherlands. 3School of Molecular and Cellular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK. 4The Pirbright Institute, Pirbright, UK. 5National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, China. 6Diamond Light Source, Didcot, UK. 7These authors contributed equally: Daming Zhou, Yuguang Zhao. *e-mail: [email protected]; [email protected] 414 Nature MICROBIOLOGY | VOL 4 | MARCH 2019 | 414–419 | www.nature.com/naturemicrobiology NATURE MICROBIOLOGY LETTERS a b CV-A5 CV-A12 CV-A3 CV-B5 CV-B2 CV-A8 CV-B1 CV-A10 CV-A7 CV-A2 CV-A14 CV-A4 EV71 CV-A16 CV-A6 c d Fig. 1 | Phylogeny and the quality of the EV71–SCARB2 EM structure. a, Phylogenetic tree of the HFMD-causing enteroviruses derived by comparing the capsid sequences. Viruses using SCARB2, CAR and KREMEN1 as receptors are circled in red, blue and green, respectively. b, EV71–SCARB2 complex viewed down the twofold icosahedral axis, with the left half of the particle shown as a three-dimensional reconstruction coloured by radius from blue, through cyan, green and yellow, to orange from lowest to highest radius, and the right half of the complex shown as ribbons coloured in blue, green, red and orange for VP1, VP2, VP3 and SCARB2, respectively. c,d, Electron potential maps for the bound pocket-binding inhibitor NLD (magenta) and surrounding residues (c), and for residues at the EV71 (green sticks)–SCARB2 (orange sticks) interface (d). was at sufficient resolution to build and refine an atomic model by nine complex glycans, the EV71-binding site is largely unhin- of the complex (Fig. 1c,d, Methods and Supplementary Table 1). dered, although a long well-ordered phosphorylated sugar has been The viron is unexpanded, with NLD remaining bound in the VP1 seen to approach this region of the surface6 (Fig. 2a). Considering pocket (Fig. 1c and Supplementary Fig. 1b), and is essentially indis- that it is highly exposed in the apo structure, the binding site is tinguishable from the native mature virus (root mean square devia- surprisingly hydrophobic, suggesting that this region is involved tion: 0.4 Å for 774 Cα atoms matched out of 784; Supplementary in protein–protein interactions as part of its function in the host. Fig. 1c,d). The receptor interacts with the virus through helices α 5 Indeed, it has been proposed that its partner β -glucosidase uses this and α 7, which—together with α 4—form a bundle lying distal from region as part of its attachment site6 (Supplementary Fig. 2b), and the domain termini, and therefore from the membrane (Fig. 2a)6. the areas of α 5 and α 7 directly involved in viral interactions make Although this is consistent with the previous inference that the face-to-face contacts with the same region of another SCARB2 mol- C-terminal end of α 4 is involved in attachment, and the observation ecule in a SCARB2–antigen-binding fragment crystal structure8. In that α 5 forms part of the epitope recognized by an antigen-binding addition to hydrophobic interactions, there are limited hydrogen fragment, which binds SCARB2 to prevent virus attachment, the bond and charge interactions, which are described below from the binding areas on both the receptor and the virus are quite different perspective of the virus. Overall, the footprint of the receptor on from those predicted3,7,8. It has been noted that this helical bundle the virus is ~700 Å2, which is similar to that observed for tightly undergoes pH-dependent conformational changes, and it has been binding antibodies21. proposed that these are involved in the pH-dependent triggering The SCARB2-binding site on EV71 comprises residues from the of viral uncoating3,6. Interestingly, although our structural analysis VP2 EF and the VP1 GH loops, which form part of the south wall was performed at relatively low pH (5.1), the structure of the heli- of the canyon and bear antigenic residues.

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