{"paper_id":"2f5133aa-b087-41fc-b5fc-b7a54668d73f","body_text":"Enterovirus A71 adaptation to heparan sulfate comes with capsid stability tradeoff  1 \n  2 \nHan Kang Tee 1*, Gregory Mathez 1, Valeria Cagno 1, #Aleksandar Antanasijevic 2, #Sophie 3 \nClément1, #Caroline Tapparel 1,3* 4 \n  5 \n 6 \n1 Department of Microbiology and Molecular Medicine, University of Geneva Medical School, 7 \nGeneva, Switzerland. 8 \n2 Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne 9 \n(EPFL), CH-1015 Lausanne, Switzerland. 10 \n3 Lead contact 11 \n# These authors contributed equally 12 \n*Corresponding authors: han.tee@unige.ch; caroline.tapparel@unige.ch  13 \n 14 \nSummary 15 \nBecause of high mutation rates, viruses constantly adapt to new environments. When 16 \npropagated in cell lines, certain viruses acquire positively charged amino acids on their 17 \nsurface proteins , enabling them to utilize negatively charged heparan sulfate (HS) as an 18 \nattachment receptor. In this study, we used enterovirus A71 (EV -A71) as model and 19 \ndemonstrated that unlike the parental MP4 variant, the cell -adapted strong HS-binder MP4-20 \n97R/167G does not require acidification for uncoating and release s its genome in the 21 \nneutral or  weakly acidic environment of early endosomes. We experimentally confirmed 22 \nthat this pH -independent entry is not associated with  the use of HS as an attachment 23 \nreceptor but rather with compromised capsid stability. We then extended these findings to  24 \nanother HS -dependent strain, suggesting that adaptation to HS generally modifies capsid 25 \nstability and alters entry mechanism. Our data show EV-A71 pH-independent entry for the 26 \nfirst time and, more importantly, highlight the intricate interplay between HS -binding, 27 \ncapsid stability, and viral fitness , wherein enhanced multiplication in cell lines leads to  28 \nattenuation in hostile in vivo environments such as the gastrointestinal tract.  29 \n 30 \nKeywords: Enterovirus; heparan sulfate; uncoating; virus adaptation; virus capsid stability  31 \n 32 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nIntroduction 33 \nHeparan sulfates (HS) are linear, negatively charged polysaccharides connected to various 34 \ncell surface and extracellular matrix proteins. Expressed on a wide range of cells, they play a 35 \npivotal role in various biological processes, and many viruses exploit them to attach and 36 \nconcentrate onto cell surfaces before binding to main entry receptor 1. Despite a substantial 37 \nbody of literature on the subject, the actual implication of HS binding on viral infections 38 \nremains a topic of debate.  39 \nEnterovirus-A71 (EV-A71) is an excellent example of the ongoing controversy regarding the 40 \nimpact of HS receptor utilization in viral pathogenesis . This virus is  a member of the 41 \nPicornaviridae family and the most neurotropic EV after poliovirus. It causes significant hand, 42 \nfoot and mouth disease  outbreaks, particularly in Asia -Pacific countries, and is  associated 43 \nwith severe neurological complications , notably in small children and immuno suppressed 44 \npatients2. The virus uses human scavenger receptor class B member 2  (SCARB2) as the main 45 \nreceptor for internalization and uncoating3,4. Since SCARB2 is mostly localized on lysosomal 46 \nmembrane and sparsely on plasma membrane 3,5, it seems to  play only a minor role in EV -47 \nA71 cell attachment6. Consistently, numerous other EV A71 receptors have been described  48 \nin the literature,  including HS3,7. When propagated in cell culture, EV -A71 rapidly acquires 49 \nadaptive mutations (i.e. patches of positively charged amino acids on the viral capsid) that 50 \nallow them to bind HS, sometimes with high avidity. These strong HS -dependent variants 51 \ngrow efficiently in cell culture but show attenuated virulence in animal models, such as mice 52 \nand cynomolgus monkeys 8-11. Analysis of the  differential expression of  SCARB2 and HS i n 53 \ntissues from monkey or transgenic mice  revealed little overlap. S trong HS expression  was 54 \ndetected in sinusoidal endothelial cells and vascular endothelia, where SCARB2 was not 55 \ndetected9,10. Similarly, HS expression in the brain was mainly found in vascular endothelia 56 \nbut SCARB2 expression was found predominantly in neuronal cells. The authors of these 57 \nstudies concluded that binding to HS on endothelial cells in absence of SCARB2 leads to viral 58 \ntrapping, abortive infection, and attenuation9,10. Similar observations were shown for other 59 \nviruses, including Murray Valley encephalitis 12, Japanese encephalitis 12, Sindbis 13, Theiler’s 60 \nmurine encephalomyeliti14, tick-borne encephalitis15, West Nile16 and dengue17.  61 \nWe previously isolated cell-adapted EV-A71 mutants with strong affinity for HS which 62 \nemerged upon passaging of intermediate HS binders derived from both patient and mouse- 63 \nadapted MP4 strains in cell culture 18,19. The mutants presented two amino acid changes in 64 \nthe VP1 capsid protein: VP1-L97R mutation in the VP1 BC loop, shown to confer 65 \nintermediate affinity for HS together with a second ary mutation, VP1-E167G, located in the 66 \nVP1 EF loop, which significantly strengthened HS binding with reduction of negative 67 \ncharges19,20. As previously observed for strong HS-binding variants, we showed that,  in 68 \ncontrast to the original mouse-adapted MP4 strain which exhibited virulence in mice, this 69 \ncell-adapted MP4-97R/167G double mutant was completely attenuated in mice 19. In the 70 \ncurrent study, we used MP4 and MP4-97R/167G mutant as representatives of respectively, 71 \nweak and strong HS-binders, slow and fast-growing in cell lines and virulent and avirulent in 72 \nmouse models (as documented previously 19,20), to elucidate the consequence of virus 73 \nadaptation towards HS binding on the viral growth cycle. We demonstrated that these 74 \nmutations not only increase binding to HS, but also reduce capsid stability, leading to 75 \nimproved uncoating and faster cell internalization in a HS-independent manner. Of note, 76 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nanother strong HS binder harboring VP1-E145Q substitution also showed decreased capsid 77 \nstability compared to the wildtype HS-independent variant. These data provide another 78 \npossible explanation for the in vivo  attenuation of strong HS-binders which may originate 79 \nfrom viral trapping but also from decreased capsid stability which would be detrimental to 80 \nthe virus in challenging environments such as the gastrointestinal tract.   81 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nResults 82 \nLysosomotropic drugs reduce infectivity of the HS-independent MP4 but enhance 83 \ninfectivity of the strong HS-binder MP4-97R/167G  84 \nFirst, we sought to assess whether viruses displaying different dependence on HS exploit 85 \ndifferent growth cycle pathways. We compared the effect of lysosomotropic drugs, namely 86 \nhydroxychloroquine (HCQ) and bafilomycin A1 (BAF -A1) on MP4 and MP4-97R/167G double 87 \nmutant. As presented in Fig. 1A , Vero cells were pre -treated for 1 hour with each drug 88 \nbefore infection. T hese drugs showed no cytotoxic eff ect at the concentrations  used in the 89 \nassay (Fig. S1A ). Inoculation was then performed for 1 hour in presence of the drug and 90 \ninoculum was removed and replaced with fresh drug -free media. The number of infected 91 \ncells was determined at 24 hours post-infection (hpi) using immunofluorescence. To confirm 92 \ninhibition of endosomal acidification by these drugs, the presence or absence of acidic 93 \nlysosomes was assessed by immunostaining of  lysosomal-associated membrane protein 1 94 \n(LAMP1) and by staining with  lysotracker, a dye specific for acidic compartments. The 95 \namount of LAMP1 and lysotracker double-positive lysosomes decreased drastically following 96 \ntreatment with the drugs, confirming the inhibition of endosomal acidification ( Fig. 1B). The 97 \neffect of the drugs on viral replication was then compared for the two variants (Fig. 1C & D). 98 \nMP4 infectivity was significantly reduced by both drugs in a dose -dependent manner, while 99 \nMP4-97R/167G infectivity was in contrast enhanced. Similar results were obtained in RD  100 \ncells (Fig. S1B), indicating that drug effects are not cell -dependent. The different sensitivity 101 \nof the two variants to acidification inhibitors was more pronounced with HCQ, so we 102 \nperformed a detailed examination of the mechanism of action with this drug. 103 \nTo determine whether the effect of HCQ was related to the usage of HS as attachment 104 \nreceptor, we repeated the virus inhibitory assay using cells depleted of HS by either 105 \nheparinase digestion ( Fig. 1E) or treatment with sodium chlorate ( Fig. 1F). The dist inct 106 \nsensitivity to HCQ was reproduced regardless of the presence or absence of HS on the cell 107 \nsurface. Of note, we confirmed that as for the human strain 20, the HS-independent and HS-108 \ndependent MP4- derivatives both need SCARB2 to infect cells and cannot replicate in 109 \nSCARB2 CRISPR-Cas9 knock-out cells (Fig. S2). Altogether, these data indicate that the capsid 110 \nmutations change the sensitivity to HCQ, independent of the attachment receptor used.  111 \nMP4 enters via SCARB2-mediated and pH-dependent endocytosis, while MP4-97R/167G 112 \nutilizes an alternative SCARB2-dependent pathway. 113 \nIn our previous studies, we showed that MP4 virus uses SCARB2 as entry receptor and here 114 \nwe demonstrated that it is inhibited by HCQ. This strongly suggests that MP4 uses SCARB2 -115 \nmediated pH -dependent endocytosis for entry and uncoating as demonstrated for many 116 \nother EV-A71 variants7,21. After having excluded that HCQ effect did not affect virus binding 117 \n(Fig. 2A), we conducted single -cycle infection assay and showed that differential effect of 118 \nHCQ on MP4 and MP4-97R/167G became prominent from 4 hpi onward (Fig. 2B). To further 119 \ndissect which step of the viral growth cycle was differentially affected by the treatmen t, we 120 \nperformed a time -of-addition assay. As shown in Fig. 2C, HCQ significantly los t its effect 121 \nwhen administered later than 2 hpi, confirming that the effect occurs during the early phase 122 \nof the viral cycle. To more specifically assess whether the drug a ffects viral entry, we 123 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\ntransfected in vitro transcribed genomic RNA containing the nanoluciferase (Nluc) gene as 124 \nreporter ( Fig. 2D). RNA transfection allows to bypass receptor-mediated entry. In these 125 \nconditions, no difference was observed for both variant s, whether HCQ was present or not 126 \n(Fig. 2E). This observation also indicates that the drug does not impact genome replication. 127 \nIn contrast, infection with infectious Nluc reporter virus reproduced the differential HCQ 128 \ninhibition as observed in the original non -modified viruses ( Fig. 2F & Fig. 1D ). Taken 129 \ntogether, these data indicate that  MP4 enters via pH -dependent endocytosis, while MP4 -130 \n97R/167G entry pathway is independent on endosomal acidification.  131 \nHCQ differentially impacts MP4 and MP4-97R/167G uncoating  132 \nTo further dissect the mechanism of action of HCQ on the entry of each variant, we next 133 \nexamined the effect of the drug on viral uncoating. We generated neutral red labelled virus 134 \nstocks and performed neutral red uncoating assay as previo usly described 22. Briefly, viral 135 \nstocks were produced in presence of neutral red in the dark to induce co -encapsidation of 136 \nviral genome and neutral red insi de viral particles. P hotoactivation of neutral red  induces 137 \nthe dye to cross-link viral genomes to the capsid  and subsequent  blockade of viral 138 \nuncoating22. Accordingly, upon infection with neutral red labelled viruses, light exposure 139 \nonly inactivates viruses that have not yet completed uncoating, while virus genomes already 140 \nreleased in the cytoplasm are unaffected. This allows to precisely define the timepoint  of 141 \nviral uncoating. Cells were pre -treated with HCQ and then incubated with neutral red -142 \nlabelled viruses for 1 hr at 37˚C for infection. Light inactivation was performed at selected 143 \ntimepoints post-infection, and infected cells were quantified 24 h later by immunostaining 144 \n(Fig. 3A). In the absence of HCQ, the majority of viruses had undergone uncoating between 145 \n2 and 3 hpi for both variants (30 -70% of uncoated viruses for MP4 and 60 -80% for MP4 -146 \n97R/167G, respectively) ( Fig. 3B). In the presence of HCQ, MP4 uncoating was completely 147 \nblocked, even when photoactivation was performed at 4 hpi  (Fig 3B, left panel). In contrast, 148 \nthe uncoating rate was not inhibited  in the presence of HCQ for MP4 -97R/167G (Fig. 3B, 149 \nright panel) and the final viral yield was even increased as already observed in Fig. 1D.  150 \nTo further validate these results, we then combined fluorescent in situ hybridization (FISH) 151 \nof viral genomic RNA and immunofluorescence staining of viral capsid at early timepoints. 152 \nFull particles are characterized by colocalization of virus genomic RNA (vRNA) and viral 153 \ncapsid (as shown  in 1 hpi at 4 ˚C), while the colocalization is lost  following the uncoating 154 \nprocess (Fig. 3C). Quantification of vRNA and capsid colocalization highlighted no significant 155 \ndifference between the two variants at 2 hpi in presence or absence of HCQ ( Fig. 3E). 156 \nHowever, at 4 hpi  (prior to the initiation of replication , see Fig. 3B ), MP4  uncoating 157 \nappeared to be inhibited by HCQ, as highlighted by a decrease of empty capsids  and an 158 \nincrease of capsid/RNA colocalization in presence of the drug  (Fig. 3D & E). An opposite 159 \neffect was observed for MP4-97R/167G, with a reduced capsid/RNA colocalization in the 160 \npresence of HCQ , indicating that more viruses had undergone uncoating in the presence of 161 \nHCQ at this time point. Altogether these data indicate that MP4 -97R/167G can uncoat s at 162 \nneutral pH and that acidification is instead increasing its replicati on capacity, while MP4 163 \nneeds acidic pH to uncoat.  164 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nMP4 relies on late endosomes for uncoating, whereas MP4-97R/167G undergoes 165 \nuncoating in early endosomes 166 \nHCQ is known to inhibit endosomal acidification by accumulating in endosomes in a 167 \nprotonated form. This accumulation leads to endosomal swelling and inhibition of fusion 168 \nbetween endosomes and lysosomes within cells, as previously described 23,24 and as shown 169 \nin Fig. 4A. We thus hypothesized that the two variants could exploit different entry routes, 170 \nwhich could explain the different sensitivity to HCQ. We showed that MP4 needs acidic pH 171 \nto uncoat and is thus expected to release its RNA in late endosomes/lysosomes. In contrast, 172 \nMP4-97R/167G can uncoat in absence of pH acidification and accordingly in a non-acidic 173 \nenvironment. To test this hypothesis, we infected Vero cells transiently expressing a variant 174 \nof small GTPase Rab5a, a protein involved in the maturation of early endosomes (EE) into 175 \nlate endosomes (LE). This Rab5a -Q79L mutant is constitutively active (CA) and blocks LE 176 \nmaturation (Fig. 4B). Viral capsids of MP4 and MP4 -97R/167G were observed within EE in 177 \nboth Rab5a WT and CA -expressing cells at 0.5 hpi and 2 hpi ( Fig. S3 and 4C). However at 7 178 \nhpi, the percentage of cells stained for double stranded RNA (dsRNA), a marker of virus 179 \nreplication, was significantly reduced for MP4 in Rab5a CA -expressing cells but not for MP4 -180 \n97R/167G (Fig. 4D). This indicates that MP4-97R/167G genomes were successfully released 181 \nin the cytoplasm to undergo translation and replication, even in absence of EE fusion to LE. 182 \nConversely, a transition from EE to LE with  a gradual pH decrease  is necessary for MP4 to 183 \nrelease its genome.  184 \nVP1-L97R/E167G substitutions confer affinity for HS but decrease capsid stability 185 \nOur data highlighted that both MP4 and MP4-97R/167G enter via a SCARB2-dependent 186 \npathway, localize in early endosomes at early times post -infection but exhibit di stinct 187 \nsensitivities to HCQ, a feature independent of their differential use of HS as attachment 188 \nreceptor. We therefore speculated that the varying pH-dependency may be attributed to 189 \ndifferences in virion stability. We used DynaMut server 25 to assess the relative influence of 190 \nVP1-97R and VP1 -167G mutations on interaction networks in their respective local 191 \nenvironments (Fig. S4A) as well as  the impact on overall capsid stability ( Fig. S4B). Indeed, 192 \nVP1-L97R mutation  is predicted to reduce hydrophobic  interactions between the original 193 \nleucine residue on this position and its neighbors VP1-245Y and VP1 -246P. Further, this 194 \nresidue is in close proximity to VP1 -244K, thereby adding more positive charge to this site. 195 \nOn the other hand, VP1-E167G mutation causes a loss in hydrogen bonding capacity to VP1-196 \n165S and reduces the net negative charge. Altogether, DynaMut analysis predicted reduced 197 \ninteraction capabilities by the substituted residues and changes in electrostatic properties 198 \nwithin the region.  This is consistent with a nalyses of vibrational entropy change , indicating 199 \nthat the presence of two  mutations result s in enhanced local dynamics, which has 200 \npreviously been correlated with reduced capsid stability (Fig. S4B)26,27.  201 \nWe therefore speculated that MP4-97R/167G mutant features a lower stability and could 202 \nbypass the need for acidic pH fo r uncoating. To test this hypothesis, we subjected these 203 \nvariants to neutral and acidic conditions and assessed their virion structure using negative 204 \nstaining electron microscopy (nsEM) ( Fig. 5A & B). At acidic pH, there was no notable 205 \nalteration in the ca psid morphology of MP4, which maintained a stable particle diameter of 206 \n~31-33 nm across both pH conditions. On the other hand, for MP4 -97R/167G, both 2D 207 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nimages and 3D reconstructions highlighted a loss of density at the center of the viral 208 \nparticles, as well as an expansion in size for a subset of particles (diameter ranging from 31 209 \nto 41 nm at pH5 versus 31 to 33 nm at pH7), indicating partial virus uncoating. We then 210 \nperformed temperature sensitivity assay by heating viruses at different temperatures for 1 h 211 \nbefore inoculation on cells. Quantification of infected cells at 24 hpi further confirmed that 212 \nMP4 capsid is more resistant to higher temperature as 80% of MP4 population survived a 213 \n50°C thermal stress compared to only 50% for MP4-97R/167G ( Fig. 5C). Consistently, the 214 \npredictions of Gibbs free energy change ( ΔΔG) induced by these mutations further 215 \nsupported that both mutations induce slight destabilization of the capsid structure, 216 \nregardless of pH and temperature (Table S1).  217 \nAs MP4-97R/167G is less stable, we hypothesized that binding to SCARB2 may be sufficient 218 \nto trigger its capsid opening. We conducted a competitive experiment and compared the 219 \ninfectivity of the two variants after incubation with soluble SCARB2 (sSCARB2) at neutral pH 220 \nfor 1 hr at 37°C. We observed that MP4-97R/167G but not MP4 variant lost infectivity upon 221 \npre-exposure to sSCARB2 ( Fig. 5D ). Altogether, our results confirmed that MP4-97R/167G 222 \ncapsid is less stable and highly sensitive to thermal and acidic stresses as well as receptor 223 \nbinding, which are sufficient triggers to initiate virus capsid disruption and subsequent viral 224 \nuncoating.  225 \nResistance to HCQ and reduced capsid stability extend to other strong heparan sulfate-226 \nbinding strains.  227 \nTo check if our observations could extend to other HS-binding variants, we checked the 228 \neffect of mutation present at position VP1-145, a residue known to play a key role in 229 \nmodulating viral HS-binding capacity and in vivo virulence. EV-A71 variants (sub-genogroup 230 \nB4) with wild-type VP1 -145E is a weak HS binder and is attenuated in mice while the cell-231 \nadapted VP1-145Q variant is a strong HS-bind er and virulent in mice 11. As shown in Fig. 6A, 232 \nHCQ enhanced infectivity of VP1-145Q variant but reduced infectivity of VP1-145E variant, 233 \nconsistent with what we observed with the MP4- 97R/167G. Both temperature sensitivity 234 \n(Fig. 6B) and sSCARB2 inhibition assays (Fig 6C) also confirmed that the capsid of VP1-145E 235 \nvariant is much more stable compared to VP1-145Q variant, in line with free energy change 236 \nprediction as shown in Table S1 . These data further reinforced the observation that HS-237 \nbinding phenotype is inversely correlated with virus capsid stability.  238 \n 239 \nDiscussion 240 \nAcidic pH is an important trigger for viral uncoating and many enveloped or non-enveloped 241 \nviruses, including influenza A virus28,29, human adenovirus30, foot-and-mouth disease virus31, 242 \nSemliki forest virus 32, that enter the cell through pH-dependent endocytosis 33. Similarly, for 243 \nEV-A71, binding to SCARB2 and subsequent endosomal acidification are required for 244 \nuncoating4,7. In this study, we provide new insights on the impact of mutations in the VP1 245 \ncapsid protein leading to strong HS affinity, on the uncoating process of EV-A71. We show 246 \nthat, unlike the mouse-adapted EV-A71 MP4 strain, the MP4-97R/167G -derived double 247 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nmutant, which has a high affinity for HS, does not require acidification for uncoating and can 248 \nrelease its genome under neutral or weakly acidic environment of early endosomes.  249 \nTo demonstrate the importance of acidification on both MP4 and MP4-97R/167G variant 250 \nuncoating, we u sed two lysosomotropic drugs, namely BAF-A1 and HCQ, that increase 251 \nendosomal pH by distinct means. On one hand, BAF-A1 inhibits the vacuolar H+ ATPase (V-252 \nATPase), preventing the acidification process and thereby elevating the endosomal pH 34-38. 253 \nOn the other hand, HCQ, a less toxic derivative of the antimalarial drug chloroquine, acts as 254 \na weak base that can be protonated and trapped in the acidic environment of cellular 255 \norganelles39-41. In addition to this effect, HCQ can impact other cellular pathways, such as 256 \nautophagy, a cellular process that has been demonstrated to be induced by EV-A71 to 257 \ncreate a favorable environment for its replication 42,43. However, we show here that the 258 \ndifferential effects on MP4 and MP4-97R/167G occur during the uncoating process rather 259 \nthan in later stages of the cycle, such as virus genome replication 40. Our results thus 260 \nunderline that, despite their distinct modes of action, both HCQ and BAF-A1 influenced virus 261 \nentry through their effect on endocytic compartments, as both compounds ultimately 262 \ninhibit the reduction in endosomal pH levels. In addition, the fact that differential sensitivity 263 \nto HCQ was retained even in cells devoid of HS at their surface by treatment with 264 \nheparinase or sodium chlorate pointed out that this pH-independent mode of entry of MP4-265 \n97R/167G is not linked to the use of HS as an attachment receptor.  Interestingly, our 266 \nexperiments using the Rab5a CA to block the transition of EE to acidic LE44,45, indicate that 267 \nbinding to SCARB2  is sufficient to trigger MP4 -97R/167G genome release into the cytosol 268 \neven in the near neutral pH of the EE (pH ∼6.0 to 6.5), whereas MP4 requires the acidity of 269 \nLE and/or lysosomes (pH ∼5.0 to 5.5)  to uncoat efficiently 46. These observations led us to 270 \nhypothesize that the two  variants exhibit intrinsic differences in capsid stability. We 271 \nconducted various tests to compare how each variant reacted to heat, sSCARB2 and low pH, 272 \nand found that MP4 -97R/167G was more sensitive to all these conditions. Particularly, we 273 \nused nsEM to study the properties of viral particles and observed an expansion  of MP4-274 \n97R/167G capsid fo llowing the  exposure to pH 5 . T hese data, plus the virus structural 275 \ndynamics prediction and free energy change computation , all indicate that MP4-97R/167G 276 \npresents reduced capsid stability compared to MP4.  277 \nOne strength of our study lies in the fact that we were able to extend these results to 278 \nanother HS-dependent EV-A71 strain, VP1-145Q variant. Given that these two HS-279 \ndependent variants share the characteristic of having incorporated a less acidic amino acid 280 \nwithin the VP1 capsid protein, we hypothesized that an increase in positive charges within 281 \nthe capsid not only enhances affinity for HS but also alters capsid stability, consequently 282 \nimpacting the virus entry mechanism. In the same line, a thermostable EV-A71 variant (VP1-283 \nK162E, change of a basic to an acidic residue) isolated from serial passages at higher 284 \ntemperatures was shown to be less efficient at uncoating with poorer cell infectivity  but 285 \nmore virulent in mice 47. Interestingly, this variant showed a more expanded conformation 286 \ncompared to the original non -mutated virus47. While the thermostable variant showed no 287 \ndifference in bi nding to SCARB2  receptor, the binding affinity to heparin was greatly 288 \nreduced, an observation consistent with what we noticed for MP4. Additional experiments 289 \nwith cell-adapted, HS-binding viruses will help to define whether HS -binding is always 290 \nassociated with a loss of virion stability and whether these findings could event extend to 291 \nother group of viruses . Interestingly, mutations conferring similar in vitro phenotypes were 292 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nobserved for other enteroviruses such as rhinovirus A16 (RV-A16) and coxsackievirus B3 (CV-293 \nB3). For RV-A16, capsid mutations conferring resistance to endosomal acidification 294 \ninhibitors also abrogated the need for acidic pH for uncoating . More importantly, these 295 \nmutations were also associated with higher sensitivity to low pH, high  temperatures, and 296 \nbinding to soluble receptors48. For CV-B3, a fast-growing variant was shown to exhibit faster 297 \ngenome release and destabilized capsid, and this led to attenuated virulence in mice 49. This 298 \nexisting literature aligns with  our findings, suggesting that alterations in capsid stability due 299 \nto amino acid changes may significantly impact various aspects of the virion and its life cycle, 300 \nincluding sensitivity to environmental factors, receptor interactions, and infection rates.  301 \nIn the light of these published studies and our data, we propose the following model 302 \ndepicting the relationship between HS -binding, capsid stability and viral fitness in vitro and 303 \nin vivo (schematized in Fig. 7). Viruses undergo mutations and positive selection to adapt to 304 \ndifferent environments50,51. EV-A71 can take advantage of the high pla sticity of its capsid to 305 \noptimize its fitness upon environmental changes. Many strains adapt to use HS in vitro due 306 \nto the abundant expression of this attachment receptor in cell lines. To do so, they usually 307 \nacquire additional positively charged amino aci ds within outward -facing VP1 domains 308 \nproximal to the capsid 5 -fold axis. In addition to help the virus to attach on the cell surface 309 \nand find the SCARB2, we show here that these mutations concomitantly decrease virion 310 \nstability. This further contributes to higher multiplication in cell lines by triggering uncoating 311 \nrapidly after internalisation, within EE, without the need of acidic pH. Interestingly in our 312 \nexperiments, acidification inhibitors improved rather than inhibited viral fitness of MP4-313 \n97R/167G. Although additional experiments are required to define the mechanism behind 314 \nthis observation, it  could occur via the protection of virions that have not yet uncoated 315 \nwhen endosome s fuse with lysosome s. In this context, absence of acidification would 316 \nimprove the chance of  these virions to release their genome in the cytoplasm, while 317 \nexposure to acidic pH would induce viral opening within the late endosomes. The situation 318 \nmay differ significantly in vivo  as strong HS binders are attenuated. Koike and colleague s 319 \nhave demonstrated that strong binding to HS induces virus trapping in vivo 9-11,19. Our data 320 \nsuggest that the associated decreased stability may further contribute to viral attenuation. 321 \nTo be virulent in vivo, a non-enveloped virus must have a sufficiently stable capsid to resist 322 \nunfavourable environmental conditions, both during dissemination within a host and during 323 \ntransmission between hosts. This last point is particularly important for EV -A71, which is 324 \ntransmitted via the fecal -oral route and must therefore resist the acidic pH of the 325 \ngastrointestinal tract before reaching the intestinal mucosa, its main multiplication  site. 326 \nCapsid stability thus ensures that virus genome release occurs only in a proper environment, 327 \nbut in turn renders the virus dependent on both SCARB2 and acidic pH for uncoating4,7.  328 \nOf note, although strong HS-binder are clearly attenuated in mice, the situation in humans is 329 \nmore puzzling as strains with Q or G at  position VP1-145 have been associated with severe 330 \nneurological cases and the methodology used in those studies excluded emergence of 331 \nmutation during cell culture 52,53. Furthermore, we previously showed that intermediate HS -332 \nbinding affinity can result in increased virulence even in mouse models 19. It is still unclear if 333 \nthere is a lack of trapping and/or limited impact on viral stability under these conditions. To 334 \nconclude, to reach optimal fitness in v itro and in vivo , the virus needs to find the correct 335 \nbalance between HS binding and capsid stability (Fig. 7 ). Our study improves the current 336 \nknowledge on the mechanism behind the in vivo attenuation of cell culture-adapted viruses. 337 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nIt also opens the doors to new antiviral strategies targeting endosomal acidification as well 338 \nnew principles for vaccine design based on attenuated -acid -independent variants to help 339 \ncombat EV-A71.  340 \n 341 \nMaterials and methods 342 \nChemical reagents 343 \nChemical reagents used in this study were listed as following: hydroxychloroquine (Tocris), 344 \nbafilomycin-A1 (InvivoGen), sodium chlorate ( NaClO3, Sigma -Aldrich), neutral red (Sigma 345 \nAldrich) and LysoTracker Deep Red (Thermo Fisher Scientific). 346 \nCell lines and virus 347 \nVero (monkey kidney; ATCC CCL-81) and human rhabdomyosarcoma cells (RD; ATCC no.: 348 \nCCL-136) were propagated in Dulbecco’s Modified Eagle Medium (DMEM) and GlutaMAX 349 \n(31966021, Thermo Fisher Scientific)  containing 10% fetal bovine serum (FBS). RD -SCARB2-350 \nKO20 and RD-ΔEXT1+hSCARB254 cells were maintained in DMEM supplemented with 10 351 \nµg/ml puromycin ( 58-58-2, Invivo Gen). All infected cells were maintained in media 352 \nsupplemented with 2.5% FBS. All cells were maintained at 37˚C in 5% CO 2. Viruses used in 353 \nthis study including MP4, MP4 -97R/167G ( Genbank accession number: JN544419 ; 354 \nsubgenogroup C2), IEQ (EV -A71 VP1-145Q variant; Genbank accession number: AF316321; 355 \nsubgenogroup B4 ) and IEE (EV -A71 VP1 -145E variant) strains were prepared as previousl y 356 \ndescribed11,19. For Nluc reporter virus, Nluc  gene was inserted between 5’ UTR and VP4 of 357 \nthe virus as previously described 55. Viruses were generated in RD-ΔEXT1+hSCARB2 cells54, 358 \npropagated for an additional passage and used as working stocks. All virus stocks were 359 \nsequenced for confirmation (Fasteris) prior to experiments.  360 \nPlasmids 361 \nPlasmids encoding eGFP-Rab5a and eGFP-Rab5a Q79L are kind gifts from Pierre-Yves Lozach 362 \n(University Claude Bernard Lyon 1).  Both IEQ and IEE plasmids ( Genbank accession number: 363 \nJN544419: AF316321; subgenogroup B4 ) strains are kind gifts from Yoke Fun Chan 364 \n(University of Malaya). 365 \nVirus inhibitory assay and time-of-addition assay 366 \nFor virus inhibitory assay, cells were pre-treated either with drugs for 1 hour at 37 ˚C. 367 \nViruses (MOI 0.1) were inoculated onto cells in presence of drugs for 1 hour at 37  ˚C. Upon 368 \ninfection, inocula were removed and cells were rinsed thoroughly with phosphate buffered 369 \nsaline buffer (PBS) before incubated with fresh media up to 24 hpi. For time -of-addition 370 \nassay, HCQ was either pretreated ( -1hpi), introduced during virus infection (0hpi) or 371 \nintroduced onto cells at post -infection (1, 2 and 3hpi) for 1hr. After incubation, cells were 372 \nrinsed with PBS before loaded with maintenance media and incubate up to 24 hpi.  For both 373 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nassays, infected cells were fixed for immunofluorescence staining for virus-positiv e cells 374 \ndetection. 375 \nVirus binding and replication assay 376 \nAll the experiments were done on Vero cells seeded in 96 wells plate. For virus binding assay, 377 \ncells were incubated with 1 × 108 RNA copy number/ml virus for 1 hour at 4  ˚C. The inocula 378 \nwere removed an d rinsed with cold PBS twice, and then subjected to cell lysis for RNA 379 \nextraction and qRT -PCR quantitation. For virus replication assay, cell monolayers were 380 \nincubated with virus for 1 hour at 37  ˚C. The inocula were removed, rinsed with PBS, and 381 \nthen furt her incubated up to 24 hpi at 37  ˚C. Infected cells were lysed and viral RNA was 382 \nquantified by qRT-PCR. 383 \nHeparan sulfate removal 384 \nBoth enzymatic and chemical methods, heparinase assay and sodium chlorate (NaClO 3) 385 \ntreatment respectively, were used to cleave H S from cell surface. For heparinase assay, the 386 \ncells were first rinsed with PBS and then incubated with 3.5 mIU/ml of heparinase III 387 \n(AmsBio) diluted in 0.1 M sodium acetate pH 7.0, 1 mM calcium acetate and 0.2% BSA for 1 388 \nhour at 37 ˚C. Meanwhile, mock -treated cells were incubated with heparinase buffer. Upon 389 \nincubation, cells were washed twice prior to virus infection. For NaClO 3 treatment, cells 390 \nwere propagated in presence of 30 mM NaClO3 at least one passage before experiment. The 391 \ncells were also pre -seeded in media supplemented with NaClO 3 and incubated overnight 392 \nbefore the experiment. 393 \nImmunofluorescence and confocal imaging 394 \nTo detect infected cells, cells were fixed with absolute methanol (Sigma Aldrich) at room 395 \ntemperature for 10 min and then incubated  with blocking buffer consisting of 5% BSA 396 \n(PanReac Applichem) and 0.05% TritonX -100 (PanReac Applichem) for 20 min. Fixed cells 397 \nwere first incubated with anti -EV-A71 capsid monoclonal antibody MAB979 (1:1000; Sigma) 398 \nfor 1 hour at 37  ˚C and then with Alexa Fluor 488-conjugated secondary antibodies (1:2000; 399 \nThermo Fisher Scientific) dissolved in DAPI solution for 1 hour at  37˚C. To detect dsRNA, 400 \ninfected cells were fixed with 4% paraformaldehyde (Santa Cruz) and incubated with 401 \nblocking buffer, cells were inc ubated with anti -dsRNA monoclonal antibody J2 (1:500; 402 \nScicons) for 1 hour at 37  ˚C and then with Alexa Fluor 594 -conjugated secondary antibodies 403 \n(1:2000; Thermo Fisher Scientific) dissolved in DAPI solution for 1 hour at  37˚C. Stained cells 404 \nwere acquired using ImageXpress Pico (Molecular Devices) and percentages of positive cells 405 \nwere determined using CellReporterXpress software. For confocal imaging, 406 \nimmunofluorescence staining was performed with EE and lysosomes were stained  using 407 \nanti-EEA1 (1:100; Santa Cruz) and anti -LAMP1 (1:100; Cell Signalling), respectively, for 1 408 \nhour at 37  ˚C and then with Alexa Fluor 488 -conjugated secondary antibodies (1:200) 409 \ndissolved in DAPI solution for 1 hour at  37˚C. The stained slides were mou nted under 410 \ncoverslip (Hecht Assistent) with Fluoromount G mounting medium (Southern Biotech) and 411 \nanalyzed using Zeiss LSM 800 confocal microscopy.  412 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nRNAscope FISH detection and colocalization experiments  413 \nFor FISH, cells were seeded on Nunc LabTek II chamber slides (Thermo Fisher Scientific) and 414 \nfixed with 4% paraformaldehyde. To detect viral RNA in infected cells, fixed cells were 415 \nprocessed for RNAscope FISH using RNAscope Multiplex Fluorescent V2 assay (Biotechne) 416 \naccording to manufacturer’s protocol. In b rief, the cells were hybridized with V -EV71-C1 417 \nprobe (Biotechne) at 40°C for 2 hours and then the signals were revealed using TSA Vivid 418 \n570 kit (Tocris). The slides were then incubated with blocking buffer and incubated with 419 \nMAB979 (1:100) at 4 °C overnight, followed by incubation with Alexa Fluor 488 -conjugated 420 \nsecondary antibodies (1:200) at room temperature for 30min. After incubated with DAPI for 421 \n30s, the stained slides were mounted under coverslip with mounting medium and analyzed 422 \nusing Zeiss LSM 800 confocal microscopy. Images were acquired and analysed using ZEN 3.2 423 \nsoftware. 424 \nLuciferase assay 425 \nEnterovirus-A71 nanoluciferase (Nluc) reporter particles were used to study virus replication 426 \nbypassing cell entry in presence and absence of drug. Briefly, the r eporter virus plasmid was 427 \nlinearized and in vitro  transcribed to generate RNA using T7 RiboMax Express Large Scale 428 \nRNA Production System (Promega). Transcribed RNA was purified using RNeasy Mini Kit 429 \n(Qiagen) and then transfected in RD cells using Lipofecta mine 2000 (Thermo Fisher 430 \nScientific). At certain timepoints, cell supernatants were harvested for luciferase activity 431 \ndetection using Nano-Glo Luciferase Assay System kit (Promega) on Glomax Multi-Detection 432 \nSystem (Promega). 433 \nNeutral red uncoating assay 434 \nTo generate neutral red (NR)-labelled viruses, virus stocks were propagated in cells in 435 \npresence of 5 µg/ml neutral red (Aldrich). The virus stocks were harvested at 3 dpi and 436 \ntitered. For uncoating assay, NR -labelled viruses were infected at 37  ˚C for 1 hr i n the dark 437 \nthen washed twice with PBS and loaded with FluoroBrite DMEM (Thermo Fisher Scientific) 438 \nsupplemented with 2.5% FBS. At certain timepoints, infected cells were exposed to light for 439 \n30 min and then allowed to incubate up to 24 hpi. Infected cells w ere analysed using 440 \nimmunofluorescence as stated earlier. 441 \nVirus infection in Rab5a-transfected cells 442 \nVero cells (1.5 × 106) were transfected with 25µ g of Rab5a-eGFP plasmids using 443 \nLipofectamine 3000 (Thermo Fisher Scientific). The next day, transfected cells  were 444 \nharvested, resuspended in buffer ( PBS, 2nM EDTA, 1% BSA ), and subjected to fluorescence -445 \nactivated flow cytometry (FACS) on S3 Cell Sorter (Biorad). EGFP -positive cells were sorted, 446 \ncollected and then further propagated at least one day before virus i nfection. For virus 447 \ninfection, cells were infected with virus (MOI 1.5) for 1 hour at 37 ˚C. The inocula were 448 \nremoved, rinsed with PBS, and cells were further incubated up to 7hpi at 37  ˚C. Cells were 449 \nthen stained with anti-dsRNA as described above.  450 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nTemperature sensitivity assay and shSCARB2 inhibition assay 451 \nViruses (MOI 0.5) were incubated at different temperatures (4°C, 37°C, 45°C, 50°C and 55°C) 452 \nfor 1hr. Upon incubation, viruses were immediately transferred onto ice for cooling down 453 \nbefore inoculated onto cells for 1hr at 37 °C. Cells were washed and allowed to incubate in 454 \nmaintenance media up to 24hpi before virus -positive cell detection using 455 \nimmunofluorescence. For SCARB2 inhibition assay, viruses were incubated with 1 µg of 456 \nsoluble recombinant human SCARB2 -FC chimera protein (bio -techne) at 37 °C for 1hr. The 457 \nmixture was then inoculated onto cells at 37 °C for 1hr. Upon incubation, cells were was hed, 458 \nand allowed to incubate in maintenance media up to 7hpi before lysed the cells for viral 459 \nRNA quantitation. 460 \nElectron microscopy (EM) 461 \nFor structural analyses, virus stocks were first inactivated by formaldehyde treatment. 462 \nFormaldehyde at 100 µg/ml final  concentration was added to the vir us stock and incubated 463 \nat 37°C for 3 days. Inactivated viruses were purified through 30% sucrose cushion at 32,000 464 \nrpm in SW32 Ti rotor (Beckman Coulter) for 14 hr at 4˚C, followed by sedimentation through 465 \na discontinuous 20-45% (w/v) sucrose at SW41 Ti rotor (Beckman Coulter) for 12 hr at 4 ˚C. 466 \nThe purified stocks were then subjected to HiPrep 16/60 Sephacryl S -500 HR column (Sigma 467 \nAldrich) with 25 mM Tris -HCl + 150 mM NaCl (pH 7.5) as the running buffer. Fractions 468 \ncorresponding to EV A71 particles were pooled and concentrated to 0.3 – 1.1 mg/mL using 469 \nAmicon Ultra centrifugal filter units with 100 kDa cutoff (Millipore Sigma). For pH -based 470 \nassays we prepared Tris -Acetate-based buffers at pH 5 and pH 7.5. The buffers compris ed 471 \n150 mM NaCl and a 100 mM mix of Tris base and acetic acid at the ratio necessary to reach 472 \nthe desired pH. Each EV A71 variant was diluted to 100 µg/ml in the two buffer and 473 \nincubated for 30 minutes. Following incubation, the samples were applied onto ne gative 474 \nstain EM grids (Cat # CF300 -Cu-50, Electron Microscopy Sciences). Prior to sample 475 \napplication the grids were glow discharged for 30  seconds. 2% solution of uranyl formate 476 \nwas used for staining. The grids were imaged on a Talos L120C G2 microscope (T hermo 477 \nFisher Scientific) running at 120 kV and featuring the CETA 4k camera. EPU software from 478 \nThermo Fisher Scientific was used for data acquisition, and all data processing was 479 \nperformed in the cryoSPARC package 56. Each dataset comprised 100 -200 micrographs, and 480 \n2’000-10’000 virus-corresponding particles. Particles were extracted from micrographs and 481 \nsubjected to 2D classification. 3D reconstruction was performed using Ab initio al gorithm 482 \nwith icosahedral symmetry imposed. 483 \nComputational analysis of virus capsid protein structure stability 484 \nTo assess the virus capsid protein structure stability, EV-A71 crystal structures with PDB ID 485 \nof 3J22 and 4AED were used for MP4 and VP1 -145 variants, respectively. I -mutant 2.0 486 \nserver57 was used to predict the free energy stability change upon introduction of mutation 487 \ninto virus capsid VP1 protein. Visualization of mutational effects on interatomic interactions 488 \nand prediction of molecule flexibility were performed on DynaMut server25.   489 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nSchematic diagram and statistical analysis 490 \nAll schematic diagrams and illustrations were created via BioRender.com. All data and 491 \nstatistical analyses were generated using GraphPad Prism 9.  All drug treatment experiments 492 \nwere analy zed with one -way and two -way ANOVA. For dose -dependent inhibitory assay, 493 \narea under curve (AUC) was calculated and analyzed using one -way ANOVA. Degree of 494 \ncolocalization of virus capsid and vRNA in individual cells was measured  using Mander’s 495 \noverlap coefficient calculation in ZEN 3.2 software. Data were presented as mean ± SEM. *p 496 \n< 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. and not significant (n.s.). 497 \n 498 \nAcknowledgement 499 \nThis work was funded in part by the Swiss national foundation (Grant N° 310030_184777 500 \nto CT) and by the University of Geneva (Salary to HKT). We would like to thank Prof Satoshi 501 \nKoike and Dr Kyousuke Kobayashi from  Tokyo Metropolitan Institute of Medical Science, 502 \nJapan for providing RD-ΔEXT1+hSCARB2 cells, Prof Jen-Ren Wang from National Cheng Kung 503 \nUniversity, Taiwan for providing infectious clone plasmids EV -A71/MP4, Prof Pierre-Yves 504 \nLozach from Université Claude Bernard Lyon 1  for providing plasmids encoding eGFP-Rab5a 505 \nand eGFP-Rab5a Q79L, Prof Yoke Fun Chan from University o f Malaya for providing IEQ and 506 \nIEE infectious clone plasmids. We would also like to acknowledge Jessica Swanson, Dr 507 \nNatalie Kingston and Prof Nicola Stonehouse for giving advice and guidance about virus 508 \npurification. Electron microscopy data was collected at the  Interdisciplinary Centre for 509 \nElectron Microscopy  (CIME) at EPFL with assistance from Davide Demurtas, PhD.  Electron 510 \nmicroscopy data was processed using the computational infrastructure provided by the IT 511 \ndepartment of the School of Life Sciences (SV -IT) at EPFL. The authors express sincere 512 \ngratitude to the CIME and SV-IT personnel for their contribution. 513 \n 514 \nConflict of interest 515 \nThe authors declare that they have no conflict of interest. 516 \n 517 \nFigure legends 518 \nFig 1. Lysosomotropic drugs inhibit infection by MP4 but not by MP4-97R/167G. (A) 519 \nSchematic illustration of the virus inhibitory assay workflow. Cells were pre -treated with 520 \nlysosomotropic drugs and infected in presence of the drug.  After inoculum removal, 521 \ninfected cells were cultured in drug -free media and infected cells were stained by 522 \nimmunofluorescence (IF) with anti -VP2 Ab.  ( B) Inhibition of endosomal acidification 523 \nconfirmed with lysotracker staining (red).  Lysosomes are in green (anti -LAMP1 Ab) and 524 \nnuclei in blue (DAPI). Representative IF images ar e shown (scale bar, 10 µm). C) Dose 525 \nresponse assay in i nfected Vero cells. Results are shown as % of virus -positive cells relative 526 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nto nontreated control. Statistical significance (one -way ANOVA) between treated and 527 \nuntreated virus or between treated MP4 an d MP4-97R/167G was calculated based on the 528 \nAUC. (D) Representative IF staining of EV -A71 (anti -VP2 in green) 24 hpi of Vero cells in 529 \npresence of 25 µM HCQ or 250 nM BAF -A1 (scale bar, 300 µm).  (E & F ) HCQ effect in Vero 530 \ncells pre-treated or not with heparinase III (hepIII) (E) or sodium chlorate (NaClO3) as in A (F). 531 \nStatistical significance (two -way ANOVA) was calculated for each virus between each 532 \ncondition. In D to F, mean and S.E.M of biological triplicates are shown. *p < 0.05, **p < 533 \n0.01, ***p < 0.001, ****p < 0.0001.  534 \nFig 2. HCQ targets viral entry. (A) Virus binding assay in Vero cells in presence of 25 µM HCQ. 535 \n(B) Single -cycle replication kinetic in nontreated and HCQ -treated Vero cells. At each 536 \ntimepoint, cell lysates were collected, and viral RNA copy numbers were quantitated using 537 \nRT-qPCR (C) Time -of-addition assay in Vero cells treated with HCQ starting at different 538 \ntimepoints. Infected cells were quantitated 24 hpi by IF . (D) Schematic diagram of Vero cells 539 \npre-treated with HCQ  and subsequently subjected to transfection of in vitro RNA transcript 540 \nor infection with EV-A71 nanoluciferase (Nluc) reporter viruses. At the indicated timepoints, 541 \ncell supernatants were collected, and luciferase activity was measured.  (E & F ) Results are 542 \nexpressed in % relative light unit (RLU) of treated versus nontreated virus at indicated 543 \ntimepoints. The mean and S.E.M from biological triplicates are shown. Statistical significance 544 \nwas calculated using two -way ANOVA, comparing treated and untreated control. *p <0.05, 545 \n**p < 0.01, ***p < 0.001, ****p < 0.0001. 546 \nFig 3. HCQ delays the uncoating of MP4. (A)  Schematic illustration of the neutral red assay 547 \nworkflow. Vero cells were pre -treated with or without HCQ for 1hr. Neutral red -labelled 548 \nviruses were allowe d for cell infection at 37°C for 1hr. Upon infection, the inoculum was 549 \nremoved and replaced with fresh media. Infected cells were exposed to light for 30 min at 550 \ndifferent timepoints and further incubated up to 24hpi for IF staining (B) Effect of light 551 \ninactivation on replication of neutral red -labelled MP4 (left panel) or MP4 -97R/167G (right 552 \npanel). Results are plotted as % of virus -positive cells relative to non -treated dark control . 553 \nMean and S.E.M of biological triplicates are shown.  Statistical significa nces (two -way 554 \nANOVA) were calculated between treated and nontreated conditions.  (C) Schematic 555 \nillustration of virus uncoating monitored with the combinational use of RNA -FISH to detect 556 \nEV-A71 RNA (red) and IF with anti -VP2 Ab to detect the viral capsid (green). Co -staining 557 \nhighlights intact viruses in yellow while empty capsids and free RNA are in green and red, 558 \nrespectively. Representative images (scale, 20 µm) of MP4 and MP4 -97R/167G binding after 559 \n1hr at 4°C (C, right panel) and of vRNA (red) and capsids (green) with and without HCQ 560 \ntreatment at 4hpi ( D). Arrows: empty capsid . (E) Co-localization of capsid and vRNA in 561 \nindividual cells at 2 hpi and 4 hpi analysed using Mander’s overlap coefficient (n = 32 562 \nindividual cells from two independent experiments). Statistical comparison (unpaired t-test) 563 \nof untreated and treated groups.  *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 564 \nFig 4. MP4-97R/167G uncoats from early endosomes. (A) Nontreated and HCQ-treated 565 \nVero cells were stained with anti-EEA-1 antibody (green) to label early endosomes, and DAPI 566 \n(blue) to label cell nuclei. (B) Schematic representation of endosomal route upon 567 \noverexpression of Rab5a WT or CA mutant. (C) Viral capsids (anti-VP2 Ab) localise in early 568 \nendosomes, 2 hpi of Vero cells transiently expressing Rab5a (in red) WT and CA. (D) % of 569 \ncells stained positive with the anti-dsRNA J2 Ab in FACS-sorted Rab5a-eGFP-expressing cells 570 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\n7 hpi. Results and statistical significance (two-way ANOVA) are expressed relative to Rab5a 571 \nWT-expressing cells. Mean and S.E.M from triplicates are shown. ***p < 0.001. In B and D, 572 \nwhite boxes are enlarged in the right panel. Scale bar: 20 µm.   573 \nFig 5. MP4 displayed stronger capsid stability and reduced sensitivity to acidification and 574 \nhigh temperatures. (A) nsEM analysis of MP4 and MP4 -97R/167G incubated at pH7 and 575 \npH5. Representative raw micrographs are shown in each case. ( B) Representative 2D class 576 \naverages generated from datasets shown in  panel A (box size = 54nm; left) and the overlay 577 \nof the corresponding 3D maps (right). Grey and orange shade indicates virus particle 578 \nreconstructions at pH7 and pH5, respectively.  (C) Temperature sensitivity assay. Infected 579 \nVero cells were quantitated by i mmunostaining with an anti -VP2 Ab at 24 hpi after 1 hr 580 \nincubation at increasing temperatures. Results are shown as % of virus -positive cells relative 581 \nto 4°C treated control. Error bars indicate mean and S.E.M from biological triplicates. (D) For 582 \nsSCARB2 in hibition assay, viruses were incubated 1h at 37°C with 1 µg of soluble SCARB2 583 \n(sSCARB2) before infection of Vero cells. I nfected Vero cells were quantitated by 584 \nimmunostaining with an anti-VP2 Ab at 24 hpi. Results are shown as % of virus -positive cells 585 \nrelative to nontreated controls. Statistically significance was calculated with two -way 586 \nANOVA. ***p < 0.001, ****p < 0.0001.  587 \nFig 6. Heparan-sulfate-binding VP1-145Q variant exhibits resistance to HCQ and higher 588 \nsensitivity to sSCARB2 inhibition and thermal stress. (A) Virus inhibitory assay with VP1-145 589 \nvariants were performed with 25 µg HCQ on Vero cells. (B) For temperature sensitivity 590 \nassays, VP1 -145 variants were incubated at increasing temperature for 1hr before 591 \ninoculated onto Vero cells. (C) For sSCARB2 inhibition assay, VP1 -145 variants were 592 \nincubated 1 h at 37°C with 1 µg of soluble SCARB2 (sSCARB2) before infection of Vero cells. 593 \nInfected cells were quantitated by immunostaining with anti -VP2 Ab at 24 hpi. Results are 594 \nshown as % of virus -positive cells relative to nontreated control (A & C) or 4°C treated 595 \ncontrol (B). Mean and S.E.M of biological triplicates are shown.  Statistically significant 596 \ndifferences (two-way ANOVA) are shown. **p < 0.01, ***p < 0.001, ****p < 0.0001. 597 \nFig 7. Seesaw model depicting the interplay between capsid mutations, heparan sulfate-598 \nbinding, capsid stability as well as the resulting fitness changes in both in vitro and in vivo 599 \nsettings. Viruses undergo continuous mutations to optimize fitness across diverse 600 \nenvironments. In cell culture, they adapt to attain an ' in vitro  advantage' by decreasing 601 \ncapsid stability while acquiring HS-binding capacity, consequently enhancing their infectivity. 602 \nConversely, during human infection, viruses adapt to secure an ' in vivo  advantage' by 603 \nbolstering capsid stability, relinquishing HS -binding capacity, and thereby evading viral 604 \ntrapping and resisting environmental stresses. 605 \nFig S1. Lysosomotropic drugs nontoxic dose-range and differential inhibition of HS-606 \ndependent and independent variants. (A) Cytotoxicity effect of lysosomotropic drugs 607 \nevaluated with LDH and MTT assays. RD and Vero cells were treated with a range of 608 \ndifferent concentrations of HCQ or BAF -A1 for 2 hr. At 24 hours post -treatment, cell 609 \nsupernatants and lysates were collected for  LDH assay and MTT assay, respectively, to 610 \ndetermine cytotoxicity effect (n =2). (B) Dose response assay with HCQ and BAF -A1 on RD 611 \ncells were performed exactly like in Vero cells ( Fig.1). Infected cells (stained with anti -VP2 612 \nAb) were quantitated at 24 hpi after treatment with increasing drug concentrations. Results 613 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nare shown as % of virus-positive cells relative to nontreated control. AUC was calculated and 614 \nstatistical significance (one -way ANOVA) between treated and untreated virus or between 615 \ntreated MP4 and MP4 -97R/167G are shown.  Mean and S.E.M of biological triplicates are 616 \nshown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 617 \nFig S2. Both EV-A71 variants are strictly dependent on SCARB2 for infection. Virus infection 618 \nwas performed on RD WT and RD  ΔSCARB2 cells. Cells were lysed, and viral RNA copy 619 \nnumbers were quantitated at 24 hpi using RT -qPCR. Results are expressed as % Virus RNA 620 \ncopy number relative to RD WT cells (set to 100%). M ean and S.E.M of biological triplicates 621 \nare shown. ****p < 0.0001. 622 \nFig S3. Viruses were detected in early endosomes at 30 mpi. Vero cells transiently 623 \nexpressing Rab5a -eGFP were infected with MP4 and MP4 -97R/167G and fixed at 30 mpi. 624 \nColocalization of viruses was imaged with Rab5a in green and virus capsid (VP2) in red.  625 \nMagnified area was highlighted in white box and displayed at left bottom of merged image. 626 \nFig S4. Visual presentation of DynaMut prediction of virus mutations on capsid amino acid 627 \ninteractions and protein stability. Prediction of changes in  amino acid int eractions and 628 \ncapsid stability induced by the VP1 -L97R and VP1 -E167G capsid mutations were performed 629 \nusing crystal structure of full assembled capsid on DynaMut server. ( A) Interatomic 630 \ninteractions displayed and compared between WT and mutant capsid struct ures. VP1 -97 631 \nand VP1-167 residues are labelled in light green and represented as sticks together with the 632 \nsurrounding interaction residues. Changes in interactions are highlighted on both WT and 633 \nmutant structures with red asterisks (*) ( B) VP1 -L97R and VP1 -E167G mutations decrease 634 \ncapsid stability.  Computation of the vibrational entropy change (ΔΔS Vib) between WT and 635 \nmutants. Amino acids in red indicate an increase of molecule flexibility.   636 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nTable S1: 637 \n 638 \nMutation pH Temperature (°C) Predicted free \nGibbs energy \nchange value \n(ΔΔG) \nVP1-L97R  7 25 -0.65 \n(Destabilizing) \n5 25 -0.70 \n(Destabilizing) \n7 55 -0.51 \n(Destabilizing) \nVP1-E167G   7 25 -1.15 \n(Destabilizing) \n5 25 -1.10 \n(Destabilizing) \n7 55 -0.74 \n(Destabilizing) \nVP1-E145Q   7 25 -0.80 \n(Destabilizing) \n5 25 -0.89 \n(Destabilizing) \n7 55 -0.71 \n(Destabilizing) \n 639 \nPredicted Gibbs free energy change value (ΔΔG) was computed using I-mutant 2 server with 640 \ncalculation formula and indication of protein structure stabilization as shown below. 641 \n 642 \nPredicted Gibbs free energy change value (ΔΔG): ΔG (new protein) - ΔG (WT) in kcal/mol. 643 \nΔΔG < 0: destabilizing mutation 644 \nΔΔG > 0: Stabilizing mutation  645 \n646 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nReferences 647 \n1. Cagno, V., Tseligka, E.D., Jones, S.T., and Tapparel, C. (2019). Heparan Sulfate 648 \nProteoglycans and Viral Attachment: True Receptors or Adaptation Bias? Viruses 11. 649 \n10.3390/v11070596. 650 \n2. Tee, H.K., Zainol, M.I., Sam, I.C., and Chan, Y.F. (2021). Recent advances in the 651 \nunderstanding of enterovirus A71 infection: a focus on neuropathogenesis. Expert 652 \nRev Anti Infect Ther 19, 733-747. 10.1080/14787210.2021.1851194. 653 \n3. Kobayashi, K., and Koike, S. (2020). Cellular receptors for enterovirus A71. J Biomed 654 \nSci 27, 23. 10.1186/s12929-020-0615-9. 655 \n4. Yamayoshi, S., Ohka, S., Fujii, K., and Koike, S. (2013). 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Nucleic Acids Res 33, 838 \nW306-310. 10.1093/nar/gki375. 839 \n 840 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 1 \nA \nPre-treatment  \nwith drug \nInfection with drug \nfor 1 hr \nImmunofluorescence \ndetection \nB \nNontreated 25 µM HCQ 250 nM BAF-A1 \nE F \nD \nNonntreated 25 µM HCQ \nMP4 MP4-97R/167G \n250 nM BAF-A1 \nC \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 2 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 3 \nInfection with/  \nwithout HCQ \nLight inactivation Immunofluorescence  \ndetection \nA B \nC \nE \n1 hpi at 4˚C  \nMP4-97R/167G \nMP4 \nD Virus capsid vRNA \n Merge \nMP4 MP4/97R/167G \nHCQ Nontreated HCQ Nontreated \nPretreatment with HCQ \nVirus infection at  \n37°C with  HCQ \nVirus –positive cell \ndetection \n-1      0      1      2     3     4                         24 hpi \nLight inactivation \nWash and replace with \nfresh media \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 4 \n .CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 5 \nA \npH 7.5 pH 5 \nMP4-97R/167G \nParticle diameter range: 31-33nm \nParticle diameter range: 31-41nm \npH 5 \n pH 7.5 \nMP4 \nParticle diameter range: 31-33nm \nParticle diameter range: 31-33nm \nMP4 MP4-97R/167G \npH7.5 pH5 \nB \nC \n D \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig 6 \n .CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nCapsid stability \nFig 7 \nHS binding affinity \nIn vivo advantage In vitro advantage \nResist to acidic pH,  \ntemperature... \npH-independent entry  \nFacilitate uncoating upon \nSCARB2 binding \n \nAvoid trapping by \nheparan sulfate \n→ Improve dissemination and transmission  → Improve multiplication  \nBinding to heparan sulfate \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig S1 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig S2 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig S3 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nFig S4 \nA \nHydrogen bonds \nWater-mediated hydrogen bonds \nWeak hydrogen bonds \nWater-mediated weak hydrogen bonds \nHalogen bonds \nIonic interactions \nMetal complex interactions \nAromatic contacts \nHydrophobic contacts \nCarbonyl contacts \nB \nVP1-L97R VP1-E167G \nΔΔSVib ENCoM: 0.055 kcal.mol-1.K-1  \n(Increase of molecule flexibility)  \nΔΔSVib ENCoM: 0.278 kcal.mol-1.K-1  \n(Increase of molecule flexibility)  \nGLU167 \nSER165 \nLEU169 \nARG85 \nTRP171 \n TRP171 \nARG85 \nGLY167 \nGLN172 \nSER165 \nLEU169 \nARG97 \nPRO246 \nTYR245 \nLYS244 \nGLY99 \nLEU95 \nLEU95 \nLEU97 \nPRO246 \nTYR245 \nLYS244 \nGLY99 \nSER243 \nWT L97R/167G \nVP1-167 VP1-97 \n* * \n* \n* \n* * \n* * \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint \n\nTable S1 \nMutation pH Temperature (°C) Predicted free \nGibbs energy \nchange value \n(ΔΔG) \nVP1-L97R  7 25 -0.65 \n(Destabilizing) \n5 25 -0.70 \n(Destabilizing) \n7 55 -0.51 \n(Destabilizing) \nVP1-E167G   7 25 -1.15 \n(Destabilizing) \n5 25 -1.10 \n(Destabilizing) \n7 55 -0.74 \n(Destabilizing) \n7 25 -0.80 \n(Destabilizing) \nVP1-E145Q   \n5 25 -0.89 \n(Destabilizing) \n7 55 -0.71 \n(Destabilizing) \nPredicted Gibbs free energy change value (ΔΔG) was computed using I-mutant 2 server with calculation \nformula and indication of protein structure stabilization as shown below. \n \nPredicted Gibbs free energy change value (ΔΔG): ΔG (new protein) - ΔG (WT) in kcal/mol. \nΔΔG < 0: destabilizing mutation \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 25, 2024. ; https://doi.org/10.1101/2024.02.23.581741doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}