# Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex

Daniel L Hurdiss, Priscila El Kazzi, Lisa Bauer, Nicolas Papageorgiou, Francois Ferron, Tim Donselaar, Arno L W van Vliet, Bruno Canard, Etienne Decroly, Andrea Brancale, et al.

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Daniel L Hurdiss, Priscila El Kazzi, Lisa Bauer, Nicolas Papageorgiou, Francois Ferron, et al. Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex. 2021. hal-03411664

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1 Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and
2 stabilizes the hexameric complex

3 Daniel L. Hurdiss¹,²=#, Priscila El Kazzi³=, Lisa Bauer¹^=, Nicolas Papageorgiou³,

4 François P. Ferron³, Tim Donselaar¹, Arno L.W. van Vliet¹, Bruno Canard³,

5 Etienne Decroly³, Andrea Brancale⁴, Tzviya Zeev-Ben-Mordehai², Friedrich Förster²,

6 Frank J.M van Kuppeveld¹*#, Bruno Coutard⁵*#

7 ¹Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health

8 Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, The Netherlands.

9 ²Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry,

10 Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

11 ³Aix Marseille Université, CNRS, AFMB UMR 7257, Marseille, France

12 ⁴School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff

13 CF10 3NB, United Kingdom

14 ⁵Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207), Marseille, France.

15 ^Current address: Department of Viroscience, Erasmus Medical Center, 3015CA Rotterdam, The

16 Netherlands

17 =These authors contributed equally

18 *These authors share senior authorship.

19 #Corresponding authors: Daniel Hurdiss (d.l.hurdiss@uu.nl), Frank van Kuppeveld

20 (f.j.m.vankuppeveld@uu.nl) and Bruno Coutard (bruno.coutard@univ-amu.fr)

21

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**Abstract**

The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

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## 46 Introduction

Viruses belonging to the genus *Enterovirus*, within the family *Picornaviridae*, are responsible for a broad range of diseases. This genus contains four clinically relevant enterovirus (EV) species (EV-A-D) and three rhinovirus species (RV-A-C). Notable members of these species include polioviruses, coxsackieviruses, echoviruses and numbered EVs (e.g. EV-A71 and EV-D68). EV-associated diseases include respiratory infections, hand-foot-and-mouth disease, conjunctivitis, viral myocarditis, pancreatitis, aseptic meningitis, encephalitis, and acute flaccid paralysis¹. RVs cause the common cold but can also trigger the exacerbation of asthma and chronic obstructive pulmonary disease². While most EV infections are mild and self-limiting, fatal complications may arise in immunocompromised patients and young children³. Outbreaks of EV-A71 and EV-D68 represent major public health concerns because these viruses are associated with severe neurological complications⁴⁻⁶. Vaccines are only available for poliovirus and EV-A71, with the latter only approved in China⁷,⁸. Treatment of other clinically important EVs is currently limited to supportive care. Given the large numbers of serotypes (>100 EVs and >200 RVs), the development of a pan-EV and RV vaccine seems unfeasible. This underscores the urgent clinical need for the development of broad-spectrum antivirals to treat EV-associated diseases.

The EV genome encodes seven non-structural proteins (2A-2C and 3A-3D), which are involved in replication, and four structural proteins (VP1-VP4) which form the capsid. Several direct acting agents (DAA) targeting either the capsid, the viral polymerase (3D<sup>pol</sup>) or the viral protease (3C<sup>pro</sup>) have been identified. While some of these compounds reached clinical trials, their development was stopped due to limited efficacy, poor bioavailability or toxicity issues⁹. An alternative target for antiviral drug discovery is the non-structural protein 2C which exhibits several important hallmarks of a promising DAA target. 2C is functionally indispensable and plays an essential role in several steps in the EV life cycle, e.g. viral RNA replication and encapsidation¹⁰. Moreover, 2C is highly conserved¹¹, raising the possibility for broad-spectrum DAA development.

2C belongs to the helicase superfamily 3 (SF3) and acts as an ATP-dependent RNA helicase and ATP-independent RNA remodeler¹². The role of this enzyme in viral replication likely requires an oligomeric organization, which has already been observed
for recombinant 2C proteins<sup>13-15</sup>. Previous studies suggested that the functional
oligomerization state of 2C is hexameric, consistent with other members of the SF3
helicase family<sup>16,17</sup>. Structural and functional characterization of the full-length
oligomeric 2C protein remains challenging due to the N-terminal amphipathic helix
which renders the protein poorly soluble<sup>13-15,18,19</sup>. Recent structures of a soluble,
monomeric fragment of 2C from EV-A71 and PV provided molecular details of the
adenosine triphosphatase (ATPase) domain, a cysteine-rich zinc finger and a carboxyl-
terminal helical domain<sup>16,17</sup>. Nevertheless, a structure of the oligomeric 2C remains
elusive.

Given the central role of 2C during viral replication, it is not surprising that several
compounds targeting 2C, such as guanidine hydrochloride (GuaHCl), HBB, MRL-1237
and TBZE-029, have been identified<sup>18,20-22</sup>. Additionally, several drug-repurposing
screens have uncovered FDA-approved drugs such as fluoxetine, dibucaine, pirlindole
and zuclopenthixol as inhibitors of EV-B and EV-D species, but their mode of action is
not yet understood<sup>23,24</sup>. Fluoxetine (Prozac®) is a selective serotonin-reuptake inhibitor
that is used clinically for the treatment of major depression and anxiety disorders<sup>25</sup>.
Mode-of-action studies revealed that only the S-enantiomer of fluoxetine potently binds
2C and inhibits EV-B and D replication<sup>26,27</sup>. Despite the growing number of identified
2C-targeting compounds, the molecular basis for their antiviral activity remains
unknown<sup>28,29</sup>. Such information would provide an invaluable resource for structure-
based design of potent, pan-enterovirus antivirals with low toxicity.

Here, we present the 1.5 Å resolution crystal structure of the soluble fragment of
CV-B3 2C in complex with SFX. The structure reveals a highly conserved hydrophobic
pocket, distal to the ATP binding site, into which the SFX trifluoro-phenoxy moiety
inserts. To functionally validate our structure and study the inhibitory mechanism of
SFX and other 2C targeting compounds, we engineered a CV-B3 2C protein fused to
a heterologous hexamerization domain. The chimeric protein recovered ATPase
activity, allowing us to investigate the inhibitory effect of several antivirals targeting EV
2C, each of which displayed a dose-dependent ATPase inhibition. Moreover,
incubation of our engineered 2C protein with SFX or dibucaine demonstrated that
these drugs stabilize the hexameric complex. This allowed us to capture the first three-
dimensional structure of the 2C hexamer by cryo-EM and suggests an inhibitory
mechanism for these drugs. Taken together, these data provide new mechanistic insights into the mode-of-action of 2C targeting compounds and offer unique tools for the design and validation of 2C inhibitors.

## 113 Results

### 114 Crystal structure of CV-B3 Δ116-2C in complex with SFX

Inspired by recent structural reports of 2C protein fragments from EV-A and EV-C species¹⁶,¹⁷, we attempted a similar truncation strategy to obtain crystals of a fluoxetine-sensitive EV-B species member, namely CV-B3. Thermal denaturation experiments showed that SFX increased the melting temperatures (Tm + 1.51°C) of the CV-B3 Δ116-2C, confirming that this truncated protein can be bound by the drug (Figure S1). To understand how SFX binds to 2C, we attempted to crystallize our truncated 2C construct in the presence or absence of the drug. While we were able to obtain a high resolution (1.5 Å) structure of the complex (Table S1), no crystals were obtained for the apo 2C protein. This suggests that SFX stabilizes the 2C protein in a conformation prone to crystallization.

The overall fold of CV-B3 Δ116-2C is similar to the analogous structure from EV-A71 and poliovirus, comprising a Rossmann fold core domain, a zinc finger domain and a C-terminal helix (Figure 1A and S2). The Walker A motif is located on the loop connecting β1 and α1, forming a putative phosphate binding loop (P-loop). The Walker B motif is found between β3 and α2 while the Walker C motif, containing N223, is found at the tip of β4³⁰. Two large flexible loops located between strands β2-β3 and α2-β4 shield one side of the central sheet. Together, these loops form a hydrophobic cavity into which the SFX molecule binds (Figure 1A-B and S2). The interaction between SFX and the CV-B3 2C is mainly mediated by hydrophobic interactions involving the side chains of residues L157, P159, M175, D176, L178, C179, P182 and D186 (Figure 1B-C). The complex is also stabilized by a hydrogen bond between the amide group of the C179 main chain and the hydroxyl group of SFX. Finally, in one of our structures, SFX interacts via its amino group with the main chain of D186 (Figure 1C).

The accessibility of SFX can be associated with two main conformational differences between the structure of EV-A71 and CV-B3 2C proteins. The first is a 10° tilt of the α2 helix (Figure 1D), enlarging the binding site of SFX by moving D186 away from the methylamine group of SFX. Together with this, the Walker B-carrying loop is positioned
away from the binding pocket in the CV-B3 Δ116-2C, as illustrated by the 210° rotation of the side chain of Q180, making the cavity accessible to SFX. In addition, residues 180-182, located downstream of the Walker B motif, participate in main chain hydrogen bonding with the 224-AGSINA-229 loop, located downstream of motif C (Figure 2A). This provides direct evidence for crosstalk between the SFX binding site and the AGSINA loop which is a known hotspot for compound resistance and dependence mutations¹⁸.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Crystal structure of CV-B3 Δ116-2C in complex with SFX. (A) The C-terminal part of 2C is highlighted in orange. The Zinc-Finger domain is highlighted in blue, and the ATPase domain is highlighted in grey. Within the ATPase domain the catalytic center, comprising Walker A, Walker B and motif C, are highlighted in green. SFX is located close to the Walker B domain and is colored blue. (B) Zoomed in view of the SFX binding pocket showing the interacting residues and electron density for the bound drug. Green residues are part of the Walker B motif. (C) Protein-Ligand Interaction profile of SFX with the 2C residues. Hydrophobic interactions are shown as dashed lines (P159, M175, L178, P182 and D186). Hydrogen bonds are shown as blue lines (C179) and water mediated hydrogen bonds are colored light blue (D186). Figure generated using PLIP³¹. (D) Overlay of the 2C crystal structure of CV-B3 (grey) with the 2C crystal structure of EV-A71 (PDB: 5GRB, chain A) (blue) in complex with ATP-γ-S.</figcaption></figure>
163 **Mutational analysis of the SFX binding site.**

164 Sequence analysis of the SFX binding site revealed that all residues, except cysteine
165 at position 179, are conserved in SFX resistant and sensitive EV species (Figures 2A
166 and S3). To assess the functional consequences of alterations in the SFX binding
167 residues, we performed mutagenesis of amino acids within, or adjacent to, the drug
168 binding pocket. L157A, P158A, P159A, M175A, D176A, D176N, L178A, P182A,
169 D186A and D186N were reverse engineered into a CV-B3 infectious cDNA clone
170 containing a Renilla luciferase (CV-B3-Rluc) reporter gene. The replication efficiency
171 of these mutants was then assessed in the presence or absence of 2C inhibitors.
172 Nearly all substitutions abrogated viral RNA (vRNA) replication. Only P158A mutant,
173 which is not in direct contact with SFX, was able to replicate. This mutation did not alter
174 the sensitivity of 2C to SFX or GuaHCl (Figure 2B). We next tested the impact of
175 introducing less stringent mutations into the SFX binding site. These mutations were
176 selected using sequence information from other picornaviruses such as hepatitis A
177 virus and Aichivirus (Figure S3). Surprisingly, even the introduction of chemically
178 similar amino acids (i.e. L178I and D186E) resulted in no detectable vRNA replication
179 (Figure S4). Consistently, upon introduction of mutations into a CV-B3 infectious clone
180 without a reporter gene, no viable viruses were recovered, except for mutation P158A.
181 Notably, after independent passages of cells transfected with the P159A mutant,
182 viruses were recovered carrying a second-site compensatory mutation, namely A229V,
183 which lies in proximity to Pro159. This substitution was previously shown to confer
184 dependence to several compounds, including GuaHCl, HBB and MRL-1237¹⁸,²⁰,²²,³².
185 Virus carrying the P159A/A229V double mutation showed a small decrease in
186 sensitivity to SFX (Figure S5). Taken together, these data demonstrate that residues in
187 the SFX binding pocket are highly mutationally constrained, presumably to preserve
188 the function of 2C.

189

190 To gain more insight into the binding pocket of SFX, we raised resistant CV-B3 via a
191 clonal selection procedure as described previously²⁹. Several SFX-resistant viruses
192 were obtained, containing single (I227V), double (I227V/A229V) or triple
193 (A224V/I227V/A229V) mutations in the 224-AGSINA-229 loop (Figure S6). Mutations
194 in this loop have been previously identified in CV-B3 resistant to 2C-targeting
195 compounds¹⁸,³³. In addition, we previously demonstrated that substitutions C179Y,
196 C179F and F190L also provide resistance to SFX²⁹. Notably, these latter mutations, as
well as 224-AGSINA-229 loop mutations, provide cross-resistance against dibucaine (Fig S7). Our crystal structure of CV-B3 Δ116-2C in complex with SFX now allows us to cluster these mutations into two groups, those directly involved in SFX binding site (i.e. C179F, C179Y and F190L), and those in the 224-AGSINA-229 loop located downstream of the Walker C motif, which do not directly interact with SFX (Figure 2). The exact role of the 224-AGSINA-229 loop in conferring drug resistance, or dependence, is difficult to assess. However, its contribution to stabilization of the Walker B-containing loop in an “open conformation”, suitable for SFX binding, corroborates this motif as a credible hotspot for resistance mutations. To further demonstrate that this loop plays an important role in fluoxetine sensitivity, we transplanted the 224-AGSINA-229 loop from CV-B3 2C into the 2C protein of the EV-A71 BrCr strain, which contains 224-ASNIIV-229 and is insensitive to SFX, despite the SFX binding residues being conserved. Indeed, EV-A71 harboring the 224-AGSINA-229 loop gained sensitivity towards SFX, providing evidence that the 224-AGSINA-229 loop contributes to drug sensitivity (Figure S8).

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<figure><img src="image_3.png" /><figcaption>**Figure 2.** Mutational analysis of the SFX binding site in CV-B3 2C. (A) Schematic structure of CV-B3 2C highlighting the residues involved in SFX binding. The Weblogo represents the conservation of amino acids based on an alignment of the 2C protein from EVs which cause disease in humans. Virus sequences which were used for the alignment can be found in Supplement Part XYZ. The blue stars on top of the amino acid residues indicate an interaction with the ligand SFX. (B) The residues which interact with SFX were introduced into an infectious CV-B3 cDNA clone containing the *Renilla* luciferase reporter gene upstream of the capsid coding region (Rluc-CV-B3). In vitro transcribed RNA was transfected into cells and Renilla luciferase was used as a sensitive and quantitative read out for viral RNA replication.</figcaption></figure>

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Engineering an enzymatically active 2C hexamer to study the mechanism of action of SFX and other inhibitors.

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The 2C protein contains three canonical motifs required for nucleotide triphosphate binding and hydrolysis, Walker A, Walker B and motif C¹¹. Previous experiments demonstrated that SFX has little effect on the ATPase activity of the truncated, monomeric 2C protein of CV-B3³³. However, the optimal activity of SF3 helicases requires their ATPase domains to be arranged in a hexameric complex³⁰,³⁴,³⁵.

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Therefore, studies of truncated 2C proteins should be complemented with biochemical analysis of the enzymatically active hexameric complex. It has been demonstrated previously that the N-terminal domain of 2C is important for oligomerization and ATPase activity, but also renders the protein poorly soluble¹³,¹⁴,¹⁹. With this in mind, we attempted to purify the MBP-tagged full-length CV-B3 2C, which resulted in a heterogenous protein preparation as shown by the size exclusion chromatography gel filtration profile. This indicated that a mixture of high molecular weight 2C complexes were present, including a putative hexamer peak (Figure S9). As an alternative strategy, we sought to uncouple CV-B3 2C from the unfavorable biochemical properties of the N-terminal amphipathic helix but retain the ability to form hexamers. Fusion of N or C-terminal ‘assistant hexamer’ proteins is an established method to study AAA+ ATPases³⁶⁻³⁸. Based on the proposed hexamer model described for EV-A71 and PV 2C¹⁶,¹⁷, we hypothesized that we could fuse a hexameric parallel coiled-coil, CC-hex-D24³⁹, to the N-terminus of CV-B3 Δ116-2C via a 10 amino acid linker sequence (Figure 3A-B). This would, in principle, enhance the local concentration of the 2C protein and correctly orientate the molecules in a manner reminiscent of membrane attachment, normally facilitated by the N-terminal amphipathic helix⁴⁰. The small size of the CC-hex coiled-coil (32 amino acids) also permits the additional attachment of a cleavable His-MBP tag to promote solubility and facilitate affinity purification of the protein (Figure S10). As expected, the hexΔ116-2C elutes as a single peak which corresponds to a hexamer whereas Δ116-2C is exclusively monomeric (Figure 3C).

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We next employed the malachite green assay to assess the ATPase activity of our hexΔ116-2C, which demonstrated that the engineered protein is enzymatically active. In contrast, the monomeric protein and catalytic dead D177A mutant showed little to no ATPase activity (Figure 3D and S11). Altogether, the data show that the hexamerization is necessary to promote the 2C-dependent ATPase activity, offering a technological platform to screen or validate 2C ATPase inhibitors.

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<figure><img src="image_4.png" /><figcaption>**Figure 3.** Engineering a soluble, enzymatically active 2C hexamer. (A) Linear representation of the full-length CV-B3 2C protein (top) and the engineered hexΔ116-2C construct (bottom). The full-length 2C protein comprises an N-terminal domain (light grey), ATPase domain (blue), Walker A, B and C motifs (black), zinc finger domain (yellow) and C-terminal extension (green). In hexΔ116-2C (residues 116-329), the N-terminal domain, which contains the amphipathic helix (dark grey), is replaced by the 32-residue cc-hex coiled coil sequence (orange). (B) Schematic representation of the hexΔ116-2C construct. (C) Migration profile of the MBP tagged monomeric Δ116-2C and hexΔ116-2C by size-exclusion chromatography. (D) Comparison of the ATPase activity for the WT hexΔ116-2C, the hexΔ116-2C D177A mutant, WT Δ116-2C and Δ116-2C D177A mutant.</figcaption></figure>

Next, we investigated the effect of the fluoxetine enantiomers - SFX and RFX - on 2C-dependent ATP hydrolysis (Figure 4A). We showed that SFX inhibits ATPase activity in a dose dependent manner with an EC50 value of ~5 µM, in the same concentration range as the dissociation equilibrium constant (Kd of 9.5 µM) obtained in a binding assay²⁶. Consistent with the binding assay, RFX was markedly less efficient at
279 inhibiting ATPase activity. We also tested the inhibitory effect of dibucaine, HBB and
280 GuaHCl, known or suspected to target the 2C protein<sup>19,21,24</sup>. All of the compounds
281 reduced the ATPase activity of 2C in a dose-dependent manner, underscoring the link
282 between inhibition of ATP hydrolysis and antiviral effect (Figure 4B-C).

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<figure><img src="image_5.png" /><figcaption>**Figure 4. 2C inhibitors reduce the ATPase activity of 2C and have antiviral effect.**
(A-C) Effects of SFX and RFX (A), dibucaine and [2-(alpha-hydroxybenzyl)-benzimidazole] (HBB) (B), or guanidium hydrochloride (GuaHCl) (C) on 2C-dependent ATPase activity. (D) Replication of CV-B3 containing the A229V 2C mutation in the presence of GuaHCL, HBB, SFX or DIB (or a non-related replication inhibitor, BF, which targets a host lipid kinase). At 8 hours post-infection, cells were freeze-thawed and virus titers of lysates were determined. (E, F) Comparison of ATPase activity of WT 2C and the A229V mutant in the presence of HBB (E) and GuaHCL (F).</figcaption></figure>

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It is known that besides drug-resistance, 2C targeting compounds can induce drug-dependence mutations. In the latter scenario, the presence of the compound is required for efficient enterovirus replication<sup>18,20,32,35</sup>. The 2C mutant, A229V, has been shown to confer dependence on GuaHCl for efficient CV-B3 replication<sup>18</sup>, but the underlying mechanism is unknown. Upon testing replication of CV-B3 carrying the A229V 2C mutation, we observed efficient replication in the presence of GuaHCl and HBB, but not SFX and dibucaine (Figure 4D). When the A229V mutant was introduced into our hexΔ116-2C construct, we observed reduced specific ATPase activity compared to the WT protein (Figure 4E). Remarkably, the level of ATPase activity
observed with the WT 2C protein is fully and partly recovered in the presence of HBB and GuaHCl, respectively (Figure 4E-F). These data reveal that the A229V mutation negatively impacts the ATPase activity of 2C and that GuaHCl and HBB can restore the enzymatic activity.

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## SFX and dibucaine stabilize the 2C hexamer

Having demonstrated that SFX and dibucaine can inhibit the 2C-dependent ATPase activity, we next wanted to determine how these compounds affect the quaternary structure of 2C. Cryo-electron microscopy analysis of our untagged engineered protein (Figure S12), in the absence of any nucleotide or inhibitor, did not reveal any distinct oligomeric structures (Figure 4A). When the non-hydrolysable ATP analogue, ATP-γ-S, was added, ‘hook-shaped’ oligomers were apparent (Figure 4B). When hexΔ116-2C was incubated with SFX or dibucaine, well-defined hexamers were discernable (Figure 4C-D). Single-particle analysis of SFX bound hexamers produced distinct star-shaped 2D class averages, some of which contained an electron dense feature in the central channel (Figure 4E). Efforts to determine the cryo-EM structure of the SFX-bound 2C hexamer were hampered by strong preferred orientation, low abundance, and instability of the particles. Nevertheless, a ~12 Å resolution reconstruction of the complex was determined by employing a 30° stage tilt during data collection (Figure 4F and S13-14). The cryo-EM map corresponds to ~140 kDa of ordered molecular weight and has a diameter of ~110 Å, consistent with the dimensions of the EV-A71 2C hexamer model proposed previously¹⁶. Moreover, a cleft is present between the 2C protomers, which correlates with the expected location of the ATP binding pocket (Figure 4G). We next generated a hexameric model of the SFX-bound CV-B3 2C, based on the crystal structure of the JC polyomavirus large T antigen⁴¹, as described by Guan et al¹⁶, and fitted this as a rigid-body into our cryo-EM map. The overall agreement between the cryo-EM density and the hexameric model of CV-B3 2C supports the proposed mechanism of C-terminus mediated oligomerization. Indeed, in our crystal structures, the C-terminal helix (α6, α7) which appear to be broken in a “door knob” fashion into two helices α6 and α7, due to crystal packing, and α7 helix is in intensive contact with the Zinc binding domain of a symmetrical chain (Figure S15).

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<figure><img src="image_6.png" /><figcaption>**Figure 5.** SFX and dibucaine stabilize the 2C hexamer. (A) Cryo-electron micrographs of apo, (B) ATP-γ-S incubated, (C) dibucaine incubated or (D) SFX incubated hexΔ116-2C (scale bar = 10 nm). (E) Representative 2D class average from untilted micrographs of SFX incubated hexΔ116-2C. Weak density can be observed within the central of the 2C hexamer. (F) Surface representation of the 12 Å resolution cryo-EM map of hexΔ116-2C in complex with SFX, generated from 30° tilted cryo-EM data. The map is shown at a contour level of 0.0661 and the binding positions of SFX (green) and ATP (orange) are superposed. (G) As shown in (F) with a hexameric 2C model, generated using the crystal structure of the monomer, overlaid. For clarity, pore-loop residues 85-100 are omitted. (H) A 110° rotated view of the model shown in (G), with the map shown at a contour level of 0.059. Putative density for the linker and cc-hex is indicated.</figcaption></figure>

Taken together, these results indicate that SFX or dibucaine binding locks 2C in a hexameric state which does not permit ATP hydrolysis and, by extension, the associated functions such as helicase activity. Consistent with our cryo-EM observations, when size exclusion chromatography is performed on hexΔ116-2C in the presence of SFX, the complex exhibits an increased retention time, indicative of a reduced hydrodynamic radius (Figure S16A). In contrast, the presence of SFX did not greatly alter the elution profile of the monomeric Δ116-2C protein, except for a slightly more pronounced left shoulder peak, indicative of low-level oligomerization (Figure S16B). Therefore, it is likely that the SFX-mediated stabilization of the hexamer requires not only a high local concentration of protein, but also their correct orientation with respect to one another.
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Discussion

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Enteroviruses are globally prevalent pathogens responsible for many diseases. There is an unmet need for broad-spectrum therapeutics to treat EV infections. The EV 2C protein is an attractive target for direct-acting antivirals, given that it is a highly conserved and functionally indispensable protein, undertaking several pleotropic functions during the viral lifecycle including membrane rearrangement, RNA unwinding and genome encapsidation¹⁰. Over the last decades, several structurally disparate compounds targeting 2C, such as the FDA-approved drugs fluoxetine and dibucaine, amongst others, were identified²³,²⁴. Subsequently, a number of resistance mutations were obtained to gain insights into their mode of action³³. However, the binding sites and the mode of action remained elusive, hampering the rational-based improvement of the compounds. It has often been hypothesized that 2C inhibitors bind directly in the catalytic site. One study suggested that dibucaine analogues target the ATP binding site⁴², but no direct evidence supporting this hypothesis was presented. Recently, we obtained evidence for an allosteric binding site based on CV-B3, EV-A71 and EV-D68 mutants that were raised against a novel and highly potent 2C inhibitor²⁹. Viruses resistant to this inhibitor contained mutations in, or adjacent to, the α2 helix, which is distal to the catalytic site. Here, we provide a high-resolution crystal structure of the CV-B3 2C protein in complex with SFX. This crystal structure revealed a hydrophobic binding pocket that accommodated the ligand between the α2 helix and the Walker B domain. Our data prove that SFX indeed targets an allosteric site on 2C that is clearly distinct from the catalytic site. The obtained structure not only provides valuable insights into the mode of action of SFX but also represents a novel tool for structure-based drug design of highly potent 2C inhibitors.

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388

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391

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393

Several structural features are associated with the accessibility of SFX to bind the 2C protein of CV-B3 but not other enteroviruses. One structural key determinant allowing SFX to access the binding pocket is the 224-AGSINA-229 loop which is also a hot spot of resistance mutations. This is supported by our finding that exchange of the 224-ASNIIV-229 loop of the SFX insensitive EV-A71 BrCr with the 224-AGSINA-229 loop implemented a SFX sensitive phenotype to the resulting chimeric EV-A71 virus. Clearly, the 224-229 loop is not the only determinant for SFX sensitivity since we discovered previously that several clinical isolates of EV-A71 without changes in the 224-ASNIIV-229 are sensitive to SFX. Unfortunately, and despite extensive efforts, an
apo-structure of CV-B3 is not available to elucidate the structural changes which occur
upon SFX binding. Comparison of the SFX-bound CV-B3 2C to that of EV-A71 (apo
and ATP-γ-S-bound) and PV (apo) shows that the α2-helix is tilted 10° away from the
SFX binding site in CV-B3 2C, which creates the solvent accessible volume required
for drug binding. Similarly, the Walker B loop of CV-B3 2C is positioned further away
from the SFX binding site compared to EV-A71 and PV 2C, which further contributes
to formation of the hydrophobic pocket required to accommodate SFX. This suggests
that, in solution, 2C exhibits a high degree of conformational plasticity and that SFX
bind to and stabilizes, an "open conformation" of 2C. Ultimately, an apo structure of
CV-B3 2C is required to confirm this hypothesis.

The crystal structure of the 2C monomer in complex with SFX is not sufficient to
decipher its mode of action as this should take into account the expected hexameric
nature of AAA+ proteins. Several lines of evidence suggest that the catalytically active
form of 2C is composed of higher oligomeric structures¹²,¹³,³⁵. Production of 2C without
its N-terminal helical domain yields monomeric protein that has little, if any, ATPase
activity³³. To overcome the biochemical limitations of 2C and to study the mode of
action of 2C-targeting antivirals, we engineered an ATP hydrolyzing, hexameric form
of the 2C ATPase domain. The resulting construct produced homogenous oligomers
that displayed robust ATPase activity compared to the inactive monomeric form of 2C
(Figure 3). Using the engineered protein, we demonstrate that the FDA approved drugs
fluoxetine, dibucaine as well as GuaHCl and HBB inhibit the ATPase activity in a dose-
dependent manner. Thus, our hexameric 2C construct represents a novel tool for in
vitro screening of 2C-targeting ATPase inhibitors. This tool might be of particular
interest to identify potential 2C inhibitors for difficult-to-culture enteroviruses such as
the RV-C species members⁴³. Besides this, this platform can provide mechanistic
insights into the mode of action of specific mutations, as shown here for the drug-
dependent A229V mutation which we revealed to impair ATPase activity in the
absence of inhibitors, but to stimulate ATPase activity in the presence of GuaHCl and
HBB.

A hexameric structure of 2C has been long sought-after in the picornavirus field but
has remained elusive due to the unfavorable biochemical properties of the full-length
protein, especially its amphipathic N-terminal α-helix. Therefore, cryo-EM analysis of
CV-B3 2C protein, incubated with SFX, provides the first 3D reconstruction of a
hexameric complex, and represents a significant step towards understanding this
enigmatic protein. Although artificially generated by the addition of an hexamerization
domain, the recovery of the ATPase activity and the binding of SFX unambiguously
confirm the relevance of the construct. Previous studies have shown that the 2C protein
of foot-and-mouth disease virus (FMDV), a member of the *Aphthovirus* genus within
the picornavirdae family, employs a coordinated ATP hydrolysis mechanism. Using
negative stain analysis, the same study demonstrated that FMDV 2C(34-318),
containing the motif C mutation N207A (which interferes with ATP hydrolysis but not
binding), could form hexamers in the presence of ATP and RNA¹⁴. It is likely that
enterovirus 2C proteins must undergo conformations changes during ATP hydrolysis,
RNA binding and unwinding, to drive their helicase function. Our biochemical and cryo-
EM analysis suggests that SFX and dibucaine binding prevents propagation of these
conformational changes by locking 2C in a defined hexameric state. A similar
mechanism was shown for a small molecule inhibitor of the human AAA+ ATPase,
p97⁴⁴. The allosteric p97 inhibitor binds at the interface of two adjacent AAA domains
and prevents the conformational changes that are required for ATPase activity of p97.
As SFX does not bind to a 2C-2C interface, it's likely that drug binding induces
conformation changes within the monomer that translate to stabilization of the
hexamer. As shown for several AAA+ ATPases, cryo-EM provides insights that are not
available from crystallographic analysis alone⁴⁴⁻⁴⁶. In the case of 2C, the observed
stabilizing effect of SFX, observed in solution, complements the high-resolution
information obtained by crystallography and provides insights into the allosteric
inhibitory mechanism of 2C targeting compounds. Our findings emphasize the
importance of using an integrative structural biology approach to fully understand the
function and mechanisms of inhibition of 2C. Previous studies have shown that purified,
full-length poliovirus 2C cannot be uncoupled from its solubility tag, and contains a
mixture of oligomeric species – both of which would hamper structural analysis¹³.
Similarly, an N-terminally extended 2C protein of echovirus 30 was shown to form
heterogenous ring-shaped structures¹⁵. Here we demonstrate that replacement of the
N-terminal region of 2C with a hexamerization domain can produce a homogenous
complex amenable to cryo-EM. Going forward, our engineering strategy should
facilitate further structural and functional studies of oligomeric 2C proteins, and their
interaction with inhibitors, viral RNA, and other viral/host factors.
460 **Material and methods**

461 **Cells and reagents**

462 Buffalo Green Monkey (BGM) cells (purchased from European Cell Culture Collection [ECACC]) and HeLa R19 cells (American Type Culture Collection [ATCC]) were 463 cultured in Dulbecco's Modified Eagle Medium (DMEM; Lonza, Switzerland) 464 supplemented with 10% (vol/vol) fetal calf serum (FCS; Lonza). All cell lines were 465 grown at 37 °C in 5% CO2. Medium was refreshed every 2-4 days, and cells were 466 passaged at >80% confluence with the use of PBS and trypsin-EDTA (0.05%) for up 467 to 10 passages. The cell lines were routinely tested for mycoplasma contamination. 468 SFX, RFX, GuaHCl and dibucaine were purchased from Sigma Aldrich. HBB was 469 provided by Prof. Andrea Brancale (Cardiff University). GuaHCl and ATP-γ-S ((Sigma 470 Aldrich) were dissolved in water at 2 M and 20 mM stock concentration, respectively. 471 All other compounds were dissolved in DMSO at 10 mM stock concentration. 472

473 **Cloning and construct design**

474 For crystallography experiments, the coding sequence of CV-B3 2C (amino acids 117 475 to 329) was cloned into the expression vector pmCox 20A as previously described⁴⁷. 476 For ATPase assays and cryo-EM experiments, the coding sequence of CV-B3 2C 477 (amino acids 116 to 329) was inserted into pET-28b plasmid. The hexΔ116-2C 478 construct was produced by inserting a 32-residue codon optimized cc-hex coding 479 sequence followed by a linker (resulting peptide: GELKAIAQELKAIAKELKAIAWELKAIAQGAG; 480 linker: GSGSYFQSNA Genscript) at 481 the 5' of the CV-B3 2C coding sequence. For both constructs, a 3C protease cleavable 482 N-terminal His₆-MBP tag was included to facilitate expression and purification. An 483 overview of the hexΔ116-2C construct is shown in Figure S10. The D177A and A229V 484 variants were produced by site directed mutagenesis using the Δ116-2C and hexΔ116- 485 2C plasmids as a template. 486

486 **Protein expression and purification**

487 The CV-B3 Δ116-2C protein used for crystallogenesis was produced in *Escherichia coli* T7 Iq Express pLysS (New England BioLabs). Cells were grown in TB medium 488 containing 100 µg/mL of ampicillin and 34 µg/mL chloramphenicol at 37°C. Expression 489 was induced overnight at 17°C with 0.5 mM isopropyl -thiogalactopyranoside (IPTG). 490 The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM 491
Tris, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 0.1% Triton) complemented with 0.25 mg/mL lysozyme, 10 µg/mL DNase and 1 mM of PMSF. The bacterial sample was sonicated and centrifuged at 18000 g for 30 min 4°C. Soluble CV-B3 Δ116-2C was purified from the supernatant by immobilized metal-affinity chromatography (IMAC) on a HisTrap column (GE Healthcare) and eluted with a buffer consisting of 50 mM Tris, 300 mM NaCl, 500 mM imidazole pH 8. The eluted protein was subject to a dialysis in 50 mM Tris pH 8, 400 mM NaCl, 10 mM Imidazole, 1 mM DTT (dithiothreitol), followed by an overnight cleavage by TEV protease (molar ratio: 1/20), followed by a second IMAC to remove the tag and uncleaved protein. The recombinant protein was finally purified by size-exclusion chromatography (SEC) on a HiLoad 16/60 Superdex 75 column (GE Healthcare) in 10 mM Hepes pH 7.5 and 300 mM NaCl.

For electron microscopy and functional assays, *Escherichia coli* Rosetta 2 (DE3) Cells (Sigma-Aldrich) were transformed with the plasmids coding for the WT, D177A and A229V CV-B3 2C proteins and grown in 2xYT medium containing 50 µg/mL of kanamycin and 30 µg/mL chloramphenicol at 37°C until the OD<sub>600nm</sub> reach 0.3. The temperature was then reduced to 18°C and when the OD<sub>600nm</sub> reached 0.5, protein expression was induced with 0.5 mM isopropyl -thiogalactopyranoside (IPTG). Following expression for 16 hours, the cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 0.1% Triton, 1 mM MgCl<sub>2</sub>) complemented with 0.25 mg/mL lysozyme, 10 µg/mL DNase I and 1 EDTA-free protease inhibitor cocktail. The bacterial sample was sonicated and centrifuged at 25000 g for 45 min at 4°C. The supernatant was passed through a 0.45 µM filter and incubated with 1 mL of Ni-NTA resin at 4°C for 1 hour on a roller. The beads were washed with wash I buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, 1 mM MgCl<sub>2</sub>) and wash II buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, 1 mM MgCl<sub>2</sub>) and then eluted with a buffer consisting of 50 mM Tris, 300 mM NaCl, 500 mM imidazole pH 8. For ATPase assays, the eluted proteins were concentrated to ~200 µL and purified by size-exclusion chromatography on a Superose 6 10/300 GL column (GE Healthcare) in 10 mM Hepes pH 7.5, 150 mM NaCl and 10 mM MgCl<sub>2</sub>. The protein-containing fractions were pooled, concentrated to ~4 mg/mL and flash-frozen in 20 µL aliquots. To remove the N-terminal His<sub>6</sub>-MBP tag for cryo-EM analysis, the Ni-NTA eluted hexΔ116-2C protein was concentrated to 200 µL, combined with 2 µg of HRV-3C protease (Sigma) and dialyzed overnight in buffer containing 50 mM Tris (pH 8),
200 mM NaCl and 0.5 mM DTT. The cleaved protein was finally purified by size-exclusion chromatography (SEC) on a Superose 6 10/300 GL column (GE Healthcare) in 25 mM Tris (pH 8), 300 mM NaCl, 1 mM MgCl₂. The fractions corresponding to hexΔ116-2C were pooled, concentrated to ~4 mg/mL and flash-frozen in 10 µL aliquots.

## Crystallogenesis, data collection and structure determination

Structure Screens 1 and 2 and Stura Footprint Screen (Molecular Dimensions Ltd) were used for initial screening. Trials were assessed in SWISSCI 3 Lens Crystallization Plates with three wells per reservoir using 400, 300 and 200 nL drops. The drops contained increasing volumes (100, 200 and 300 nL) of protein solution at a concentration of 8-9 mg/mL in complex with or without 2.5 mM SFX (from 50 mM stock in 100% DMSO) and 100 nL mother liquor. Crystal hits were observed only in presence of SFX, in 0.1M MES pH 6.5, Polyethylene glycol (PEG) 20K 12%. Optimization of crystal-growth conditions led to the following condition: 0.1M MES pH 5.7 – 6.7, PEG 20K 7.5/15 %. Crystals appeared after 24 h. They were soaked in 25% (v/v) PEG 200 added to the mother liquor and cooled in liquid nitrogen. X-ray diffraction data were collected at European Synchrotron Radiation Facility, Grenoble (ESRF). The structure was solved by molecular replacement using Phaser with PV-2C-ΔN-3Mut structure (PDB code: 5Z3Q, D chain) as the searching model and was refined to a resolution of 1.4 Å. The crystal belongs to a space group of P2₁2₁2₁, with 1 copy of CV-B3 Δ116-2C in the asymmetric unit (ASU). In addition, crystals were also obtained in another crystallization solution containing 0.1M MES pH 6.3 – 6.7, PEG 5 000 MME 23/33 %, 0,2M ammonium sulphate. Crystals in mother liquor were soaked with 0.3 µL of 20 mM ATP overnight at 20°C. The datasets were collected at Proxima-2A-Synchrotron SOLEIL. The structure was solved by molecular replacement using Phaser with the structure of CV-B3 Δ116-2C in complex with SFX as a search model and refined to a resolution of 1.5 Å. All of the data sets were processed using XDS⁴⁸ and scaled with SCALA (Collaborative Computational Project, Number 4, 1994). The model building was performed in Coot⁴⁹, and the structures were refined using phenix.refine⁵⁰. The statistics of data collection, structure refinement and structure validation were summarized in Table S1. Figures of CV-B3 Δ116-2C structures and structural alignments were drawn by Chimera⁵¹.
560 Protein stability assay

561 To assess the folding of CV-B3 Δ116-2C and its ability to bind SFX, the thermal stability
562 of recombinant CV-B3 Δ116-2C in presence of SFX was monitored by fluorescence-
563 based thermal shift assay (TSA) using a Bio-Rad CFX Connect. TSA plates were
564 prepared by dispensing into each well the 2C protein (final concentration of 15 µM in 10
565 mM Hepes pH 7.5 and 300 mM NaCl) which was mixed with 1 µL of SFX (from 20 mM
566 stock in 100% DMSO, 1 mM final concentration in 4% DMSO) and a SYPRO orange
567 solution in concentrations recommended by the manufacturer in a final volume of
568 25 µL.

569 Reverse engineering of SFX interaction residues

570 The CV-B3 mutations in 2C L157A, P158A, P159A, M175A, M175I, M175G, D176A,
571 D176N, L178A, L178I, P182A, D186A, D186E, D186N were first introduced into the
572 Rluc-CV-B3 reporter viruses with Q5 site-directed mutagenesis kit (New England
573 Biolabs, Bioké, Leiden, The Netherlands)⁵²,⁵³. The Rluc-CV-B3 reporter viruses contain
574 a *Renilla* luciferase gene upstream of the capsid coding region which allows a fast and
575 quantitative read-out for virus replication. Next, a 705 bp fragment containing the
576 desired mutation was isolated using the enzymes BssHII and XbaI and reintroduced
577 into the original non-mutagenized Rluc-CV-B3 backbone. After site-directed
578 mutagenesis the newly generated plasmids were subjected to Sanger sequencing to
579 confirm the existence of the 2C mutation. The corresponding primers which were used
580 for side-directed mutagenesis can be found in Table S3. Viral RNA was transcribed in
581 *vitro* using the T7 RiboMAX Express Large Scale RNA production system (Promega,
582 Leiden, The Netherlands) according to the manufacturer's protocol. In a 96-well, HeLa
583 R19 cells were transfected with 7,5 ng of viral RNA. One hour after transfection, the
584 medium was replaced by fresh medium and/or compound containing medium. After 8
585 hours, cells were lysed and luciferase activity was determined using the Renilla
586 luciferase Assay System (Promega, Leiden, The Netherlands). Since most of the
587 mutations abrogated virus replication, we cloned the above-mentioned 2C mutations
588 into the p53CB3/T7 infectious clone using BssHII and XbaI⁵³. Similar to the Rluc-CV-
589 B3 plasmids, the infectious clones were linearized with MluI and RNA was transcribed
590 *in vitro*, and 150 ng of viral RNA was transfected into 6-wells containing either HeLa
591 R19 or BGM cells. All transfections were done in triplicates. Transfection of CV-B3-
592 2C[A229V] was done in the presence of 1mM GuaHCl. 3 days after transfection, the
transfected cells were subjected to three freeze-thaw cycles and the lysates were
passaged 3 times on either HelaR19 or BGM cells, respectively. If CPE was observed
during passaging of the viruses, viral RNA was isolated with the NucleoSpin RNA Virus
kit (Macherey-Nagel, Leiden, The Netherlands) according to the manufacturer's
protocol. The viral RNA was reverse transcribed into cDNA with random hexamer
primers using the TaqMan Reverse Transcription Reagents (Applied Biosystems) and
PCR was performed to isolate the 2C regions with the forwards primer binding in 2B 5'
CTAACCAAATATGTGAGC 3' and the reverse primer binding in 3A 5'
CTCACTGTCTACCGATTTGAG 3'. The same primers were used for Sanger
sequencing of the PCR product. If virus was obtained, virus titers were determined by
endpoint dilution titration and calculated according to the method of Reed and Muench
and expressed as 50% cell culture infective dose (CCID₅₀)⁵⁴. The obtained CV-B3-
2C[A229V] virus was titrated in the presence of 2mM GuaHCl.

Single-cycle infection

Confluent HeLa R19 (25.000 cells/ well in a 96-well) cells were infected with virus at a multiplicity of infection (MOI) of 0.1 or 1 at 37 °C for 30 min. Next, the medium was removed, and fresh (compound-containing) medium was added to the cells. At the indicated time points, the medium was discarded, and cells were subjected to three times freeze thawing to determine the virus titers by endpoint dilution using the methods of Reed and Muench. In the case of RLuc-CV-B3 infection, cells were lysed 8 hrs post infection to determine the luciferase activity with the *Renilla* luciferase Assay System (Promega). The CV-B3-2C[A229V] mutant was titrated in the presence of 2mM GuaHCl.

ATPase Assay

The release of inorganic phosphate during 2C-mediated ATPase hydrolysis was
measured using the Malachite Green Phosphate Assay Kit (Sigma Aldrich,
Zwijndrecht, The Netherlands). Each 50 µL reaction comprised 500 nM recombinant
2C protein and 1 mM ATP in reaction buffer (10 mM HEPES (pH 7.5), 150 mM NaCl
and 10 mM MgCl₂). The reaction buffer was always prepared fresh and recombinant
2C proteins were thawed immediately prior to use. For testing the inhibitory effect of
2C targeting compounds, the final DMSO concentration in each reaction was 1%.
Samples were incubated for 30 min at 37°C and then diluted 4-fold with water to give
a final ATP concentration of 0.25 mM, as per the manufacturer's instructions. To terminate the enzyme reaction, 80 µL of the diluted sample was mixed with 20 µL of Solution AB in a 96-well plate. The plates were incubated for 30 min at room temperature and absorbance was measured using a microplate reader at OD<sub>630</sub>. To determine the phosphate concentration in each sample, the OD<sub>630</sub> values were plotted against a standard curve using GraphPad Prism version 8.

## Cryo-electron microscopy

HexΔ116-2C (1 mg/mL) was either used directly for grid preparation or first incubated with 5 mM ATP-y-S, 100 µM SFX or 100 µM dibucaine (final DMSO concentration of 0.5%), at 4°C for 30 minutes. 3 µL of each sample was dispensed on Quantifoil R1.2/1.3 200-mesh grids (Quantifoil Micro Tools GmbH) that had been freshly glow discharged for 30 seconds at 20 mA using a PELCO easyGLow™ Glow Discharge Cleaning System (Tedpella). Grids were blotted for five seconds using Whatman No. 1 filter paper and immediately plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV plunger (Thermo Fisher Scientific) equilibrated to ~95% relative humidity, 4°C. Screening of the apo, ATP-y-S incubated and SFX or dibucaine incubated samples was performed using a 200 kV Talos Arctica (Thermo Fisher Scientific) equipped with a Gatan K2 Summit direct detector. Subsequently, two data sets were collected on the SFX incubated sample using a Titan Krios Cryo-TEM (Thermo Fisher Scientific) operating at 300 keV. For the first data set, movies were collected using a K2 direct electron detector operating in electron counting mode, at 165,000× magnification corresponding to a calibrated pixel size of 0.842 Å/pix over a defocus range of -1.5 to -2.5 µm. 1527 movies were collected using a dose rate of 8.8 e-/pix/sec for a total of 4 seconds (40 fractions), resulting in a total exposure of ~50 e-/Å² (1.25 e-/Å²/fraction). To account for the preferred orientation exhibited by the 2C hexamers, an alpha tilt of +30 degrees was used for the second data collection. Movies were collected using a K3 direct electron detector operating in super resolution mode, at 64,000× magnification corresponding to a super resolution pixel size of 0.69 Å/pix over a defocus range of -2 to -4 µm. 6119 movies were collected using a dose rate of 12.6 e-/pix/sec for a total of 2 seconds (37 fractions), resulting in a total exposure of ~54 e-/Å² (1.45 e-/Å²/fraction). All cryo-EM data were acquired using the EPU 2 software (Thermo Fisher Scientific).
657 Image processing

658 For the untilted data, collected movie stacks were manually inspected and then 659 imported in Relion version 3.0.1<sup>55</sup>. Drift and gain correction were performed with 660 MotionCor2<sup>56</sup>, and GCTF was used to estimate the contrast transfer function for each 661 micrograph<sup>57</sup>. Approximately 200 particles were picked manually and 2D classified. 662 The best resulting class was then used as a template for autopicking in Relion, 663 resulting in 42,206 particles. Fourier binned (2 × 2) particles were extracted in a 240- 664 pixel box and subjected to a round of 2D classification after which 3616 particles were 665 retained. Using the ‘molmap’ command in UCSF chimera, residues 358-628 of the JC 666 polyomavirus helicase (PDB ID: 5J40<sup>41</sup>) were used to generate a 50 Å resolution 667 starting model. Particles selected from 2D classification were 3D auto-refined (with C6 668 symmetry), which produced a highly anisotropic map. Following unsuccessful attempts 669 to solve the preferred particle orientation with buffer additives, tilted data collection was 670 employed as described previously<sup>58</sup>. Tilted movies were Fourier binned (2 × 2) during 671 motion correction with MotionCor2<sup>56</sup>, and goCTF was used to estimate the defocus 672 gradient of each micrograph<sup>59</sup>. Approximately 1000 particles were picked manually and 673 2D classified. The best resulting class was then used as a template for autopicking in 674 Relion, resulting in 180,069 particles. Fourier binned (2 × 2) particles were extracted 675 in an 80-pixel box and subjected 3D classification, after which 53,854 particles were 676 selected. After a second round of 3D classification, 11,024 particles were retained. A 677 final round of no-alignment 3D classification was performed, yielding a final stack of 678 6856 particles. Subsequent 3D auto-refinement (with C6 symmetry) and post- 679 processing yielded a map with an estimated resolution of 12 Å, based on the gold- 680 standard FSC = 0.143 criterion. An ad-hoc negative B-factor of –200 Å<sup>2</sup> was applied 681 during the final post-processing step. An overview of the data processing pipeline is 682 shown in Supplementary Figure 13. Figures of the CV-B3 hexΔ116-2C reconstruction 683 were made in UCSF ChimeraX<sup>60</sup>.

684 Data availability

685 Atomic coordinates and structure factors for CV-B3 Δ116-2C in complex with SFX are 686 deposited in the Protein Data Bank under accession codes 6S3A and 6T3W. The EM 687 density map for the SFX incubated hexΔ116-2C has been deposited to the Electron 688 Microscopy Data Bank under the accession EMD-12798. All reagents and relevant 689 data are available from the authors upon request.
690 Acknowledgments

691 DLH is funded from the European Union's Horizon 2020 research and innovation
692 program under the Marie Skłodowska-Curie grant agreement (No 842333) and holds
693 an EMBO non-stipendiary long-term Fellowship (ALTF 1172-2018). PEK is the
694 recipient of a scholarship of the Foundation "Méditerranée Infection", Marseille. This
695 work was supported by the European Union (Horizon 2020 Marie Skłodowska-Curie
696 ETN 'ANTIVIRALS', grant agreement number 642434 to AB, FJMvK and BCo). This
697 work is also supported by Netherlands Organization for Scientific Research (NWO-
698 ECHO-711.017.002 to FJMvK) and NWO-VICI-91812628 to FJMvK), and from the Life
699 Science Research Network Wales (grant no. NRNPGSep14008 to AB, an initiative
700 funded through the Welsh Government's Ser Cymru program). This work was
701 supported by the European Research Council under the European Union's
702 Horizon2020 Programme (ERC Consolidator Grant Agreement 724425 - BENDER).
703 This work benefited from access to the Netherlands Centre for Electron Nanoscopy
704 (NeCEN) at Leiden University, an Instruct-ERIC center with assistance from Dr.
705 Rebecca Dillard. We thank Dr. Mihajlo Vanevic for computational support.

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