Seneca Valley virus attachment and uncoatingmediated by its receptor anthrax toxin receptor 1Lin Caoa,b,c,d,e,1, Ran Zhange,1, Tingting Liub,1, Zixian Sune,1, Mingxu Hue, Yuna Sunf, Lingpeng Chenge, Yu Guoa,c,Sheng Fud,e, Junjie Hug, Xiangmin Lib, Chengqi Yuh, Hanyang Wangi, Huanchun Chenb, Xueming Lie, Elizabeth E. Fryj,David I. Stuartj,2, Ping Qianb,2, Zhiyong Loud,2, and Zihe Raoa,c,d,e,f

aDrug Discovery Center for Infectious Disease, College of Pharmacy, Nankai University, 300071 Tianjin, China; bState Key Laboratory of AgriculturalMicrobiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070 Hubei, China; cCollege of Life Sciences, Nankai University,300071 Tianjin, China; dMinistry of Education Key Laboratory of Protein Science, School of Medicine, Tsinghua University, 100084 Beijing, China; eSchool ofLife Sciences, Tsinghua University, 100084 Beijing, China; fNational Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science,100101 Beijing, China; gHubei Colorectal Cancer Clinical Research Center, Hubei Cancer Hospital, 430071 Wuhan, China; hTsinghua University Affiliated HighSchool, 100084 Beijing, China; iNanjing Foreign Language School, 210046 Nanjing, China; and jDivision of Structural Biology, University of Oxford,Headington, OX3 7BN Oxford, United Kingdom

Edited by Stephen C. Harrison, Boston Children’s Hospital and Harvard Medical School and Howard Hughes Medical Institute, Boston, MA, and approvedNovember 8, 2018 (received for review August 20, 2018)

Seneca Valley virus (SVV) is an oncolytic picornavirus with selec-tive tropism for neuroendocrine cancers. SVV mediates cell entryby attachment to the receptor anthrax toxin receptor 1 (ANTXR1).Here we determine atomic structures of mature SVV particlesalone and in complex with ANTXR1 in both neutral and acidicconditions, as well as empty “spent” particles in complex withANTXR1 in acidic conditions by cryoelectron microscopy. SVV en-gages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading tostructural changes in the VP1 GH loop and VP3 GH loop, whichattenuate interprotomer interactions and destabilize the capsidassembly. Despite lying on the edge of the attachment site, VP2D146 interacts with the metal ion in ANTXR1 and is required forcell entry. Though the individual substitution of most interactingresidues abolishes receptor binding and virus propagation, a serine-to-alanine mutation at VP2 S177 significantly increases SVV prolif-eration. Acidification of the SVV–ANTXR1 complex results in a majorreconfiguration of the pentameric capsid assemblies, which ro-tate ∼20° around the icosahedral fivefold axes to form a previouslyuncharacterized spent particle resembling a potential uncoating in-termediate with remarkable perforations at both two- and three-fold axes. These structures provide high-resolution snapshots ofSVV entry, highlighting opportunities for anticancer therapeuticoptimization.

Seneca Valley virus | ANTXR1 | cryo-EM | receptor recognition | entry

Seneca Valley virus (SVV) is the prototypic member of theSenecavirus genus within the Picornaviridae family (1, 2).

SVV selectively infects and lyses neuroendocrine cancer cells,including small-cell lung cancer (SCLC) and pediatric neuroen-docrine solid tumors (2–4). This is stimulating efforts to developSVV as an oncolytic agent against these tumors, which are amajor cause of morbidity and mortality (SCLC alone is respon-sible for ∼30,000 deaths annually in the United States) (5). In-deed, preclinical and early-phase clinical trials have confirmedthe safety and efficacy of SVV as a cancer treatment. Very re-cently anthrax toxin receptor 1 (ANTXR1) was identified asmediating the initial attachment of SVV to permissive cells anddownstream entry events (5).ANTXR1, also known as tumor endothelial marker 8 (TEM8),

was first identified as the cell surface receptor of anthrax toxin(6). Unlike the wide distribution in adult tissues of another an-thrax toxin receptor, ANTXR2 (also known as capillary mor-phogenesis protein 2, CMG2), ANTXR1 is abundant in tumorcells and the vasculature of developing embryos (7, 8). It wasreported that among 1,037 cell lines in the Cancer Cell LineEncyclopedia (CCLE), over 63% of cell lines exceed the ex-pression cutoff of ANTXR1 (5). ANTXR1 has an N-terminalextracellular von Willebrand factor type A (vWA) domain

containing a metal ion-dependent adhesion site (MIDAS) motifsimilar to the integrin I domain (6), connected to a C-terminalcytosolic domain by a single transmembrane-spanning domain (6).Although many different cellular factors have been identified

as entry receptors, the visualization of picornavirus–receptorinteractions at atomic or near-atomic resolution is still relativelyrare, due in some cases to the inherent flexibility of the receptorbinding portions. A number of enterovirus receptor complexeshave been determined, including: PV with CD155 (9–11), majorgroup HRV and CVA21 with intercellular adhesion molecule 1(ICAM-1), minor group HRV with very-low-density lipoproteinreceptor (VLDLR) (12–15), and CVB3 with coxsackievirus–adenovirus receptor (CAR) and decay-accelerating factor (DAF)

Significance

Seneca Valley virus (SVV) selectively infects and lyses neuro-endocrine cancer cells, including small-cell lung cancer (SCLC)and pediatric neuroendocrine solid tumors, which are a majorcause of morbidity and mortality. It is under development inclinical trials as an oncolytic agent against these tumors. Indeedpreclinical and early-phase clinical trials have confirmed thesafety and efficacy of SVV as a cancer treatment. We determinethe atomic structures for SVV–ANTXR1 in different conditions.A most attractive finding is that a serine-to-alanine mutation atVP2 177 position on the interface significantly increases pro-liferation. The molecular details for SVV–ANTXR1 attachmentand uncoating shown in this work provide clues to optimizeSVV as an oncolytic reagent with higher efficacy and lowerimmunogenicity.

Author contributions: H.C., D.I.S., P.Q., Z.L., and Z.R. designed research; L. Cao, R.Z., T.L.,Z.S., M.H., C.Y., and H.W. performed research; M.H., S.F., and Xueming Li contributed newreagents/analytic tools; L. Cao, R.Z., T.L., Z.S., M.H., Y.S., L. Cheng, Y.G., J.H., Xiangmin Li,H.C., Xueming Li, E.E.F., D.I.S., P.Q., Z.L., and Z.R. analyzed data; and H.C., E.E.F., D.I.S.,P.Q., Z.L., and Z.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The cryo-EM density maps and structures have been deposited in theProtein Data Bank, www.pdb.org, and the Electron Microscopy Data Bank (EMD) [acces-sion nos.: mature particles in pH 8, 6ADT and EMD-9613; mature particles in pH 6, 6ADSand EMD-9612; SVV(full)–ANTXR1 in pH 8, 6ADR and EMD-9611; SVV(full)–ANTXR1 in pH6, 6ADM and EMD-9608; and SVV(spent)–ANTXR1, 6ADL and EMD-9607].1L. Cao, R.Z., T.L., and Z.S. contributed equally to this work.2To whom correspondence may be addressed. Email: dave@strubi.ox.ac.uk, qianp@mail.hzau.edu.cn, or louzy@mail.tsinghua.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1814309115/-/DCSupplemental.

Published online December 4, 2018.

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(16, 17). In many cases the receptor binds in a depression on theenterovirus surface, termed the canyon, although VLDLR bindsHRV2 VP1 close to the icosahedral fivefold vertex (13, 14).Several picornaviruses bind integrin receptors, usually via aflexible arginine–glycine–aspartic acid (RGD) motif. A recentcryo-EM study captured medium resolution views of the foot-and-mouth disease virus (FMDV)–ɑvβ6 integrin complex and revealedthe attachment via a conserved RGD motif in a very exposed andflexible VP1 GH loop (18). Notably, the complex structuresreported to date have generally been determined at neutral pH(13, 18).Structures of mature SVV and the procapsid have been de-

termined previously by crystallography and cryo-EM (19, 20),and the binding of ANTXR1 to SVV has been visualized at lowresolution (5, 19). Structural comparison of the capsids suggeststhat SVV lies between aphthoviruses and cardioviruses, and isquite distant from enteroviruses (21). Little is known about theuncoating process in any picornavirus; here we throw light onthis issue by the detailed analysis of SVV–ANTXR1 interactionsand the capture of what appears to be an uncoating interme-diate, allowing us to speculate how receptor binding to SVVmight mediate genome release under acidic conditions in thelate endosome.

Results and DiscussionSample Preparation. We purified SVV mature particles at neu-tral pH (8) and, as expected, they contain genomic RNA (SIAppendix, Fig. S1). To determine the structure of the SVV–

ANTXR1 complex, these mature particles were incubated withrecombinant ANTXR1 (residues 38–220) at pH 8 for 8 h(Methods and SI Appendix, Fig. S1). Since these SVV particlescontain the viral genome, we named this complex SVV(full)–ANTXR1. Acidic conditions are known to induce capsid disso-ciation and genome release for several picornaviruses (22). Totry to understand this process in SVV, we treated mature par-ticles with ANTXR1 at pH 4.9–5.1 and found that all particlesdissociated immediately (SI Appendix, Fig. S1). We thereforerepeated the experiment at a slightly higher pH (5.9–6.1). Underthese conditions, we were able to capture a small fraction (∼10%of all observed particles) of receptor-bound spent particles[named SVV(spent)–ANTXR1 hereafter] (SI Appendix, Fig. S1).In contrast, in the absence of receptor, SVV mature particles arestable under acidic condition (pH 6) for over 4 h (SI Appendix,Fig. S1), indicating that both receptor binding and acidic con-dition are required for SVV genome release.Initial cryo-EM reconstructions were performed by using the

standard procedure in Relion (23). The local defocus of eachparticle was then further optimized in THUNDER (24), by re-fining defocus during the Expectation Maximization step. Withthe help of this strategy, the resolution of the postprocessedgold-standard Fourier shell correlation (FSC) increases from3.66 Å to 3.22 Å for the SVV mature particle at pH 8, 3.08 Å to2.84 Å for the SVV mature particle at pH 6, 3.50 Å to 3.38 Å forthe SVV(full)–ANTXR1 complex at pH 8, 3.55 Å to 2.84 Å forthe SVV(full)–ANTXR1 complex at pH 6, and 3.63 Å to 3.08 Åfor the SVV(spent)–ANTXR1 complex (SI Appendix, Fig. S2).The significant increase in resolution probably arises in part fromthe ability of the defocus determination in THUNDER to ex-clude the strong signal from the carbon film, which allowsTHUNDER to estimate the true defocus of the center of eachparticle.

SVV Mature Particles Interact with ANTXR1. To understand thestructure of the initial virus–receptor attachment complex, wedetermined the structure of SVV in complex with ANTXR1 atpH 8 (Figs. 1 and 2A and SI Appendix, Figs. S2–S4 and Table S1).The micrographs showed bound ANTXR1 and no evidence ofcapsid dissociation. The structure was determined by single

particle analysis assuming icosahedral symmetry, and the high-quality density for the receptor is consistent with rigid attach-ment at high occupancy, in line with the KD value at 49.9 nM(Fig. 2E and SI Appendix, Fig. S5). The quality of the density forthe ANTXR1 molecule, particularly in the region of the SVVbinding interface and the corresponding density for the SVVcapsid proteins, allows detailed interpretation of the virus–re-ceptor interactions (Fig. 2A and SI Appendix, Fig. S2). In thecryo-EM density of SVV(full)–ANTXR1 at pH 8, the local res-olutions of the interface and the capsid were calculated at 3.1 Åand less than 3.1 Å, respectively, while the resolution of the restof the bound ANTXR1 was calculated at 3.4 Å (SI Appendix,Fig. S4).Under neutral conditions, the architecture of the SVV mature

particles is the same as reported for SVV-001, with VP2/VP4interacting closely with the packaged genome (20). Residues inthe VP1 CD loop (R88, V93, S94, S96, S98, and G99) and VP2DF loop (D166–G179, T180, and Y182–W187) of SVV interactwith ANTXR1 (Fig. 2A and SI Appendix, Fig. S6). In addition, onthe edge of the binding site, D146 of VP2 rearranges slightly tobecome a ligand completing the MIDAS site (SI Appendix, Fig.S7, see Discussion below). The overall engagement is signifi-cantly different from other reported picornavirus–receptorcomplexes. In the FMDV–integrin and HRV2–VLDLR inter-actions, VP1 residues alone attach to the receptor (13, 18). ForPV, CV, and the major group HRVs, their receptors overlap atthe canyon-like depression on the capsid, and mainly involveresidues from the βC and the βE to ɑB loop of VP1 on the northrim, the GH loops of VP1 and VP3 on the floor, and the puff ofVP2 on the south rim (9, 11, 12, 15, 16) of the canyon. Thebinding of DAF to CVB3 is different; the DAF footprint on thecapsid is defined by two distinct areas from two neighboringprotomers, but the interface still lies in the canyon region (17).The SVV–ANTXR1 interaction is therefore currently unique inpicornavirus–receptor recognition.The binding surface on ANTXR1 is composed of residues

within the β2–β3 (T87 and R88) and ɑ2–ɑ3 (G115, G116, D117,Y119, H121, E122, E125, and R126) loops, and in ɑ4 (H154,D156, L157, F159, Y160, and E164). The ANTXR1R88 andANTXR1T87 side chains form hydrogen bonds or van der Waalscontacts with side chain atoms of VP1V93 and carbonyl oxygen ofVP2W187 (Fig. 2B). The side chains of ANTXR1R126/E125/E122/H121/Y119/D117 are stabilized by strong interactions withVP1R88/S94/S96 and VP2R183/N186/W187, with an additional hy-drogen bond formed between the amide nitrogen of ANTXR1G166and the hydroxyl group of VP2D166 (Fig. 2C). Residues ANTXR1E161/Y160/L157/H154 also form a set of contacts with VP1S98/G99 andVP2S177/L178/Y182 (Fig. 2D), while the side chain of ANTXR1F159interacts with VP2T180 (Fig. 2D). Finally for integrin receptors, whichalso contain MIDAS motifs, there is generally a specific motif (typ-ically RGD) which contains an acid residue which completes thecoordination of the metal ion of the integrin MIDAS (for instance,ɑvβ6 integrin and FMDV) (18), and indeed the ANTXR1 MIDASparticipates in binding anthrax toxin (6, 25). Despite the ANTXR1MIDAS being marginal to the binding site, as noted above, a smallmain-chain rearrangement in the vicinity of D146 of VP2 allows thisside chain to complete the Mg2+ coordination (Fig. 2A and SI Ap-pendix, Fig. S7). While the overall energetic gain from this interactionis unlikely to be very large, it is conceivable that the rear-rangement introduces strain which overall destabilizes the viruscapsid. The relevance of this interaction was investigated bysite-directed mutagenesis, and substitution of D146 by an ala-nine eliminated virus proliferation, confirming this as a signif-icant interaction (Fig. 2 F and G). We mutated a series of othercapsid residues whose side chains are involved in receptor bindingto alanine residues, rescued the mutated viruses, and testedwhether they affect virus proliferation (Fig. 2 F and G). The re-sults showed that VP2 triple mutant Y181A–Y182A–R183A and

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double mutant N186A–W187A completely eliminated SVV pro-liferation. VP1 R88A, VP1 S94A, and VP2 D166A attenuatedvirus proliferation to less than 10% of WT virus, whereas VP1S98A retained 50% viral proliferation (Fig. 2 F and G). In-terestingly, the titer of VP2 S177Amutated virus increased 17-foldcompared with WT virus. Most of the mutated viruses producedless cytopathic effect (CPE) and smaller plaque sizes, whereas theVP2 S177A mutant virus grew with greater CPE (Fig. 2G).Furthermore, substitutions of ANTXR1T87/D117/H121/E122/

E125/D156/Y160 by alanine residues completely eliminatedSVV–ANTXR1 interaction, with no detectable binding affinity

(Fig. 2E and SI Appendix, Fig. S5). Two exceptions are mutationsANTXR1R88A and ANTXR1F159A which have dissociation con-stants comparable with WT ANTXR1 (Fig. 2E and SI Appendix,Fig. S5). The minimal impact on SVV–ANTXR1 interaction ofANTXR1R88A and ANTXR1F159A mutations corresponds with theweak contacts formed by those side chains and SVV capsidproteins. Moreover, consistent with the structural observationthat MIDAS is distant from the core receptor binding region, thetreatment of ANTXR1 with EGTA to remove the bound Mg2+

ion, moderately attenuated the binding with SVV (KD = 58.8 nM)(Fig. 2E and SI Appendix, Fig. S5). These results confirmed thekey roles of residues in the virus–receptor interface.

ANTXR1 Attachment Leads to Shifts in the VP1 GH Loop and VP3 GHLoop. Although the binding of ANTXR1 under neutral condi-tions does not fundamentally alter the virus capsid, the VP1 GHloop and VP3 GH loop move significantly (Fig. 3A and SI Ap-pendix, Fig. S8 A–D). Both loops are pulled upwards from thevirus surface by ∼10 Å compared with the mature virus, wherethey pack stably on the surface, with the VP1 GH loop closelyinteracting with an adjacent VP3 GH loop in the pentamericassembly (Fig. 3B). This is reminiscent of the situation in FMDV,where the VP1 GH loop is elevated even more (∼20 Å) on re-ceptor binding. In both of these viruses the receptor harbors aMIDAS site, which attaches to the virus; however, in FMDV theattachment is almost entirely through the RGD and neighboringhydrophobic residues of the VP1 GH loop, and the VP3 GHloop displays negligible movement (18), whereas the SVV plat-form comprises a large surface area (∼1,000 Å2, comparable tothose typically seen for high-affinity antibody–antigen interac-tions) and the elevated regions are not the main components ofthe binding site. The rearrangement of the SVV VP1 GH andVP3 GH loops eliminates most contacts between them (Fig. 3C),possibly increasing the flexibility of the protomer assemblies. It isinteresting that the conformational change is seen despite theVP1 GH loop being distant from the site of SVV–ANTXR1interaction.

Fig. 1. Overall structures. The densities for SVV mature particles, spentparticles, or their complexes with ANTXR1 under neutral or acidic conditionsas determined by cryo-EM. On the Right of each image, the front half of thedensity has been cut away so that a cross-section of the binding can be seen.Depth cueing is used such that color indicates radius (<110 Å, blue; 120–150 Å,from cyan to yellow; and >160 Å, red). Clouds of red density show the boundreceptor. Icosahedral five- and threefold axes are indicated by pentagons andtriangles.

Fig. 2. Attachment of ANTXR1. (A) Structure of a protomer of the SVV–ANTXR1 complex is shown in ribbon representation. VP1, VP2, VP3, and VP4polypeptides are colored blue, green, red, and yellow, respectively. The bound ANTXR1 is drawn as orange ribbon. All structures are covered with density. Theinterface is framed and shown as a close-up view on the Right. (B–D) Close-up of SVV–ANTXR1 interface. Contacting residues are displayed as sticks with the samecolor scheme as in A. Dashed lines indicate the contact with the distance ranging from 2.4 Å to 3.6 Å. (E) Binding affinities of SVV mature particles with differentANTXR1 mutations measured by SPR. The native data are shown in SI Appendix, Fig. S5. (F) Titrations of rescued viruses with indicated mutations on the virus–receptor interface. (G) Plaques formed in BHK-21 cells by wild-type and mutant SVVs. The patterns of CPE correlated with plaque size. N.D., not detectable.

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The Effect of Low pH on the SVV Capsid, With and Without Receptor.The acidic environment in the late endosome is known to inducegenome uncoating in many picornaviruses (26–29). We deter-mined the high-resolution structure of SVV mature particles atpH 6 with final resolution 2.84 Å. The overall structures of SVVat pH 8 and pH 6 are essentially identical, except that residues93–98 in the VP1 CD loop and 145–157 in the VP2 EF loop arenot visible in the low pH map (SI Appendix, Fig. S9).Acidification of the SVV(full)–ANTXR1 complex to pH 6

resulted in two distinguishable kinds of particles (SI Appendix,Fig. S1). One of these, containing viral genome, was indistin-guishable from the pH 8 complex structure (rmsd 0.31 Å, 0.25 Å,and 0.62 Å for the Cɑ atoms of VP1, VP2, and VP3, respec-tively). The VP1 CD loop and VP2 DF loop are clearly seen inthe reconstruction from these particles, thus attachment of thereceptor prevents the disordering of these loops at pH 6, sug-gesting that their disordering at low pH is not likely to be directlyassociated with uncoating.The second type of particle observed is strikingly different. It

remains icosahedral but differs in composition, since there is nodensity corresponding to the genome (Fig. 1), and it also differsin structure, with the icosahedral pentameric units being rear-ranged compared with the mature virus, although the receptorremains attached in the same fashion (Fig. 4). In addition tolacking density for the genome, the density corresponding to the29 residues at the N-terminal of VP1, 43 residues at the N-terminal of VP2, and the entire VP4 are largely missing inthese particles (SI Appendix, Fig. S10). Previous studies showedthat the lack of an N terminus of VP1 and VP4 are features of

genome releasing intermediates of enteroviruses (28, 30) and arerelated to the lack of RNA in FMDV (31). From these charac-teristics, we propose that this spent particle most likely repre-sents the state immediately following genome release.The receptor-bound low-pH spent particle is different, not

only from the mature particle but also from the “natural empty”or procapsid of SVV, which is very similar in overall structure tothe mature virus particle, and different from the reported en-terovirus genome-releasing intermediate or expanded form (28,29, 32–35). In the spent particle, each pentameric assembly hasrotated ∼20° clockwise around the corresponding icosahedralfivefold axis (Fig. 4 A–C). Massive changes in the orientation ofthe pentameric plates within icosahedral picornavirus assembliesare not unprecedented, thus for FMDV, attempts to visualize anassembly intermediate led instead to the formation of an “inside-out” reassembled shell (36), whereas for another aphthovirus,equine rhinitis virus (ERAV), there is a clockwise rotation ofthe pentamers by more than 30° on storage in a 30% sucrose-containing buffer at 4 °C (37). It is not possible to tell whetherthe ERAV structure is a spent particle, analogous to ours, or is areassembled shell, akin to the FMDV structure. What has beenknown for some time is that, unlike enteroviruses which undergoa transition to an altered somewhat expanded form (28, 29),aphtho- and cardioviruses proceed via disassembly into pentamers(38). Since SVV appears to be intermediate between aphtho- andcardioviruses the spent structure, showing movement of wholepentamers, is in line with the type of uncoating transition expectedFig. 3. Conformational shifts of capsid proteins upon receptor binding. (A)

Protomers of SVV capsid in mature particles (white), the SVV(full)–ANTXR1complex (colored) and SVV(spent)–ANTXR1 complex (dark red) are alignedand drawn as cartoons. VP1 GH loop and VP3 GH loop are framed andshown as close-up view on the Right. ANTXR1 is not shown to clarify thepresentation. (B) Pentameric assemblies of SVV mature particles (Left) arecompared with the SVV(full)–ANTXR1 (Middle) and SVV(spent)–ANTXR1complex (Right) in the same orientation. VP1 GH loop and VP3 GH loop aremarked as colored spheres. (C) Close-up of the interaction between VP1 GHloop and VP3 GH loop in SVV mature particles (Left) compared with the SVV(full)–ANTXR1 (Middle) and SVV(spent)–ANTXR1 complex (Right). VP3 froman adjacent protomer is labeled VP3′.

Fig. 4. Spent particle. (A and B) Density for SVV(full)–ANTXR1 and SVV(spent)–ANTXR1 determined by cryo-EM drawn with radial depth cueing(<110 Å, blue; 120–150 Å, from cyan to yellow; and >160 Å, red). Icosahedralfive-, three- and twofold axes are indicated. One pentameric assembly foreach particle is outlined by a pentagon. (C) The pentamers in full (blue) andspent (red) particles are aligned with the guidance of the orientation oficosahedral axes, and rotations between them are labeled. (D and E) Threeadjacent pentamers in the capsid of SVV full and spent particles are shown ascartoons. The electron density around icosahedral twofold axes in full (F)and spent particles (G), and around icosahedral threefold axes in full (H) andspent particles (I) is shown. (J) Structural differences in VP1 (blue) and VP2(green) due to capsid rotation and expansion. The rough inner and outerboundaries of the shell are marked as gray arcs.

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and might serve as a more general model for a major set of picor-navirus genera.The tectonic movements in the spent particle significantly al-

ter the overall surface properties and the interactions that holdthe particle together are strongly reduced. In the mature particle,interactions along the interpentamer boundary are mainly be-tween VP2 and VP3 from different pentamers, with VP2–VP2interactions occurring close to the icosahedral twofold axis (Fig.4 D and F). The VP2 residues 21–27, 62–67, 95–97, 119–125,214–217, and 251–259 in one pentamer have contacts with theVP3 residues 77–82, 125–133, 145–161, and 199–207 in anotheradjacent pentamer. Meanwhile, residues 99–107 of VP2 αA he-lices from two pentamers also provide interpentamer interac-tions to stabilize the capsid. In the spent particle, the interactionsare much more tenuous, with the rotation swinging the VP2twofold interaction helix round so that it contacts VP3 instead,with the formation of mainly novel VP2–VP3 interactions (Fig. 4D and F). VP2 residues 55–59 and 97–104 interact with VP3residues 160–161 and 125–128. Moreover, VP3 residues 79–81,145–150, and 199–202 from two adjacent pentamers also contacteach other. As a result, the buried surface area of two adjacentpentamers in the spent particles is 1,150 Å2, far smaller than the3,900 Å2 interpentamer buried surface in the mature particles.Specifically, it has been reported that the αA helices of adja-

cent VP2 subunits normally form a key interaction stabilizingthe mature virus and seal a genome-releasing channel in ma-ture enteroviruses (28, 29). The VP2 αA helices are similarlyarranged in SVV mature particles (Fig. 4 D–G). In contrast inthe spent particles, instead of VP2 residues, VP3 residues 145–151 in the EF loop and residues 197–202 in the HI loop face eachother at the twofold axes but no direct interpentamer interac-tions occur (Fig. 4 D–G); indeed at the twofold axis, there is an∼10 Å × 60 Å cleft (Fig. 4 F and G) similar to the proposedgenome-releasing channel observed in expanded enteroviruses(28, 29). Also, whereas the VP2 DE/FG loops and residue 248–254, and VP3 DE/HI loops from three adjacent protomers closelycontact at the threefold axis in the mature virus (Fig. 4 D, E, H,and I), in the spent particles, the VP2 B/I/D/G strands surround alarge triangular hole, ∼45 Å along an edge, at the threefold axis(Fig. 4 D, E, H, and I).While the core structures of the individual polypeptides are

little affected by this massive change (Cαs superpose with rmsd0.52 Å, 0.66 Å, and 0.39 Å for VP1, VP2, and VP3, respectively),the icosahedral building block of the pentamer pivots a littleabout the corner of VP1 at the icosahedral fivefold axis, so thatVP3 swings outwards from the particle center by up to ∼2 Å (Fig.4J). The net effect of the rotation and pivot motion is to increase thecapsid radius by ∼2% compared with full particles (Fig. 4 A and B).It should also be noted that the VP1 GH loop and VP3 GH loopsfold back to contact the virus surface again in the SVV(spent)–ANTXR1 complex, though their conformations are different fromthose in SVV mature particles (Fig. 4 and SI Appendix, Fig. S8).

ConclusionHere we have not only determined the high-resolution cryo-EMstructure of SVV in complex with its receptor ANTXR1, but alsoobserved a snapshot of a receptor-mediated pH-triggered eventthat may represent what occurs in the late endosome at the timeof virus uncoating. In many cases inherent flexibility has limitedthe visualization of the molecular details of receptor attachmentby picornaviruses. Here high-resolution cryo-EM provided apowerful tool to visualize molecular details of the rather rigidSVV–ANTXR1 interface, including detailed information for theside chains of key interacting residues. Assisted by the VP1 CDloop, the SVV VP2 DF loop plays an essential role in bindingANTXR1. We also find that on the margins of the binding sitethe virus contributes an aspartic acid side chain (D146 of VP2)which completes the coordination shell of the Mg2+ ion of the

MIDAS site of the receptor, and abrogation of this interactionrenders the virus noninfectious. The FMDV–integrin complexstructure shows that FMDV engages integrin through comple-tion of a MIDAS site, but overall the interaction area betweenreceptor and virus is far smaller for FMDV and the receptorattachment is inherently flexible. SVV provides evidence of avirus using MIDAS recognition (via an integrin-like vWA do-main) as part of the cell attachment process in the context of arigid virus–receptor complex (18, 25). In summary, the SVV–

ANTXR1 complex expands the repertoire of receptor attach-ment sites seen in the picornavirus family and enhances ourunderstanding of the variety of receptor recognition models.The mechanism by which the RNA genome is released from

picornaviruses has long been the subject of speculation (26, 27,39, 40). Recent work suggests that for enteroviruses, expandedparticles represent the genome-releasing intermediate (28, 29),with channels close to the icosahedral twofold axes allowingegress of the genome, as well as the VP1 N terminus and VP4,which precede RNA release (28, 29). We have shown that SVValone is stable in acidic conditions and that the SVV receptorcomplex is stable at neutral pH. However, upon both receptorattachment and acidic treatment (pH 6), we find that an unusualSVV spent particle is formed, which remains attached toANTXR1. Since (i) incubation at a slightly lower pH results incomplete dissociation and (ii) these particles are found in thepresence of an excess of normal particle–receptor complexes, itseems unlikely that the spent particles are formed by reassocia-tion of dissociated pentameric assemblies. In addition to the lossof density for the genome, the VP1 N terminus, VP2 N terminus,and VP4 are also absent from this particle, as is observed inenterovirus expanded particles. The SVV particles contrastsharply with enterovirus expanded particles: the SVV pentamericassembly has a large rotation around the icosahedral fivefoldaxis. These tectonic movements lead to a remarkably large tri-angular hole, with the edge ∼45 Å, at the icosahedral threefoldaxes, together with an ∼10 Å × 60 Å cleft at the twofold axis.Combining these observations, we propose that the structuresreported here represent snapshots from the dynamic events thatoccur during the initial attachment and uncoating of SVV. Dur-ing this process, the VP1 GH loop and VP3 GH loop function assensors for the receptor attachment and could impart structuralplasticity to the capsid protomers for the formation of an un-coating intermediate (spent particles). Interestingly, the pentamerrearrangements in SVV are reminiscent of even more massivechanges seen in ERAV, which might also be related to an un-coating intermediate (37). Given that the overall structure of SVVhas greater similarity with the aphtho and cardio genera than theenteroviruses, and both aphtho- and cardioviruses uncoat toproduce pentameric capsid fragments, it seems plausible to sug-gest that what we have uncovered here for SVV may be relevantto this broad set of picornaviruses.SVV is under development in clinical trials as a therapeutic to

treat SCLC and pediatric neuroendocrine solid tumors thatcause thousands of deaths worldwide. The molecular details forSVV–ANTXR1 attachment and SVV uncoating may provideclues to help optimize SVV as an oncolytic reagent. For example,the VP2 S177A mutation at the virus–receptor interface signifi-cantly increases virus proliferation and capsid surface-exposedresidues which do not participate in receptor recognition mightbe modified to depress the immune response. Improving the ef-ficacy and lowering the immunogenicity of SVV may enhance itstherapeutic potential.

MethodsThe propagation of the SVV Hubei strain, the rescue and titration of mutatedviruses were performed as described previously (41, 42). For purification,SVV was first precipitated by the addition of 8% (wt/vol) PEG 6000 andwas then purified through a sucrose cushion followed by CsCl-gradient

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ultracentrifugation. Soluble ANXTR1 extracellular domain (residues 38–220)was prepared as previously described (25, 43). Equal volumes of virions at1 mg/mL and ANTXR1 at 5 mg/mL were mixed and kept at 4 °C overnight toproduce SVV–ANTXR1 complex. Cryo-EM data were collected at 200 kV witha FEI Arctica (Thermo Fisher Scientific) and a direct electron detector (FalconII, Thermo Fisher Scientific). Micrograph images were collected as movies(19 frames, 1.2 s) and recorded at −2.4 to −1.4 μm underfocus at a calibratedmagnification of ×110 kX, resulting in a pixel size of 0.93 Å per pixel. Indi-vidual frames from each micrograph movie were aligned and averaged us-ing MotionCor2 (44). Particles were picked and selected in Relion2.1 (23) andcontrast transfer function (CTF) parameters estimated using CTFFIND4 (45).Subsequent steps in 3D reconstruction used Relion2.1 (23) and THUNDER(24). The X-ray crystal structure of SVV-001 (PDB ID code 3CJI) and/orANTXR1 (PDB ID code 3N2N) were manually placed into the cryo-EM mapand rigid-body fitted with University of California San Francisco Chimera(46). Manual model building was performed using Coot (47) in combinationwith real space refinement with Phenix (48). The cryo-EM density maps and

structures have been deposited in the PDB and the Electron Microscopy DataBank (EMD) with accession nos: mature particles in pH 8, 6ADT and EMD-9613;mature particles in pH 6, 6ADS and EMD-9612; SVV(full)–ANTXR1 in pH 8,6ADR and EMD-9611; SVV(full)–ANTXR1 in pH 6, 6ADM and EMD-9608; andSVV(spent)–ANTXR1, 6ADL and EMD-9607. Surface plasmon resonance (SPR)analysis was carried out using a Biacore T200 (GE Healthcare). Detailed infor-mation for methods are provided in SI Appendix.

ACKNOWLEDGMENTS. We thank the computing and cryo-EM platformsof Tsinghua University, Branch of the National Center for Protein Sciences(Beijing) for providing facilities. This work was supported by NationalProgram on Key Research Projects of China Grants 2018YFD0500204,2018YFA0507200, and 2017YFC0840300; National Natural Science Founda-tion of China Grants 21572116, 81322023, 31770309, 31772749, and 31370733;Fundamental Research Funds for the Central Universities Grant 2662017PY108;and Tianjin Initiative Scientific Program Grant 10ZCKFSY08800. D.I.S. and E.E.F.are supported by the Medical Research Council (MR/N00065X/1).

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