key: cord-0982916-0c7tf0np
authors: Lang, Yifei; Li, Wentao; Li, Zeshi; Koerhuis, Danielle; van den Burg, Arthur C.S.; Rozemuller, Erik; Bosch, Berend-Jan; van Kuppeveld, Frank J.M.; Boons, Geert-Jan P.H.; Huizinga, Eric G.; van der Schaar, Hilde M.; de Groot, Raoul J.
title: Coronavirus hemagglutinin-esterase and spike proteins co-evolve for functional balance and optimal virion avidity
date: 2020-04-05
journal: bioRxiv
DOI: 10.1101/2020.04.03.003699
sha: 0c8847f9dc0c83bcb8f9f91bc75c82c44e1218c8
doc_id: 982916
cord_uid: 0c7tf0np

Human coronaviruses OC43 and HKU1 are respiratory pathogen of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spill-over. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase HE acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity towards clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked-out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor-binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations selected for cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and co-evolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses (IAVs). Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

cell culture supernatant with BCoV HE-Fc 17 to final concentrations of 1 pg to 10 μg/ml, and allowed 155 infection to proceed for 120 hr. While in the absence of HE-Fc there was no sign of virus propagation 156 as detectable by IFA, concentrations of exogenous sialate-O-acetylesterase as low as 1 ng/ml to up to frequency in p3. As a result of this mutation, HE lectin affinity was regained albeit to levels solely 207 detectable by high-sensitivity nanobead hemagglutination assay, while esterase activity towards 208 clustered glycotopes in BSM increased 4-fold as compared to HE-Phe 211 Ala, but still remained 125-fold 209 lower than that of wildtype HE (sFig. 4). Apparently, the increase in HE function, minor as it may be, 210 provides a selective advantage, but apparently one that benefits both low and high affinity S variants, 211 because the mutation was found in cloned viruses of either type. 3.4 x 10 7 TCID50/ml, when measured in the presence or absence of exogenous HE-Fc, respectively.

217 Surprisingly, in stark contrast to trial 1, the trial 2 p1 population was comprised for about 94% of 218 viruses expressing wildtype BCoV S. Less than 6% consisted of variants with mutations in S1 A , four of 219 low receptor binding affinity (Thr 22 Ile, Asn 27 Tyr, Val 29 Gly, His 173 Tyr), one of near-wildtype binding 220 affinity (Pro 174 Leu), and, with the exception of Thr 22 Ile, all at positions seen before (Tables 1, 2) . 221 Consistent with our previous findings, however, virus purification through endpoint dilution in the 222 absence of exogenous HE-Fc yielded low affinity mutants (11/11 tested) exclusively (sFig. 5). If the 223 variants in the trial 2 p1 population were all of equal replicative fitness under the conditions applied, 224 the odds of this result would be less than 1.10 -12 .

At first glance, the two trials would seem to differ in their outcomes. We offer, however, that the 226 results are in fact consonant and that the main difference is in the speed with which the virus 227 populations increased and evolved. There is an inherent stochastic element to the experimental 228 approach and whether the developing quasispecies undergoes slow track (experiment 1) or fast track 229 evolution (experiment 2) is likely dependent on the time of advent of the first mutant virus and its 230 properties, for instance whether it is an ultralow (like Ile 26 Ser) or low affinity variant (like Asn 27 Ser).

The findings allow for several conclusions. (i) They confirm and firmly establish that loss of HE lectin function selects for mutations in S1 A that reduce S receptor-binding affinity and virion avidity. (ii) The 233 possibilities to reduce the affinity of the S RBS through single site mutations are finite. In several 234 independent experiments, substitutions in S1 A occurred at a limited number of positions albeit not 235 necessarily by the same residue. For example, Asn 27 was replaced both by Ser and Tyr. (iii) The 236 mutations that reduce S affinity fall into different categories. Most map within or in close proximity of 237 the RBS to affect receptor-ligand interaction directly. Others, like His 173 Tyr and Pro 174 Leu, are more 238 distal from the RBS and apparently affect ligand binding indirectly through long range conformational 239 effects. A third type of mutations, quasi-random Cys substitutions, apparently disrupt S1 A folding by 240 promoting non-native disulfide-bonding. While an Arg 197 Cys substitution strongly decreased secretion 241 of the S1 A -Fc fusion protein, the biosynthesis and intracellular transport of native trimeric spikes was 242 seemingly affected to lesser extent. At least, the uptake of S-Arg 197 Cys into VSV pseudotypes was not 243 noticeably impaired as compared to that of wildtype S (sFig. 3E). Still, the mutation did alter the 244 infectivity of the pseudotyped particles making them less dependent on exogenous HE-Fc (sFig. 3F) 245 presumably by reducing the avidity of S trimers through local S1 A misfolding and consequential 246 disruption of the RBS in one or more monomers. (iv) Perhaps most surprisingly, quasispecies 247 developed in which loss of HE lectin function was compensated at the level of the viral population 248 with minority low affinity variants, constituting less than 6% of the swarm, not only sustaining the 249 replication of high affinity variants but actually allowing the latter to flourish and amplify to become 250 the majority phenotype.

S and HE proteins co-evolve to attain functional balance and optimal virion avidity. Among the first 252 mutations fixed upon zoonotic introduction and early emergence of OC43, was a HE-Thr 114 Asn 253 substitution, which created a glycosylation site at the rim of the lectin domain RBS 11 (Fig. 3A) . Glycans 254 attached to HE Asn 114 hamper binding to 9-O-Ac-Sia through steric hindrance, causing a 500-fold 255 reduction in HE avidity (Fig. 3B) and a 125-fold in sialate-O-acetylesterase-activity, respectively ( Fig.   256 3C). We introduced the HE Thr 114 Asn substitution in BCoV, expecting that the glycosylation site would 257 be rapidly lost through any of several single-nucleotide restorative mutations in HE. Indeed, NGS analysis of the virus swarm arising after targeted recombination showed the glycosylation site to be 259 destroyed but only in 10% of the population and exclusively by Ser 116 Phe substitution (Fig. 3D) . This 260 mutation partially restores HE receptor binding and receptor destruction to 0.125 and 0.17 of that of 261 wildtype HE, respectively (Fig. 3B, C) . In the vast majority of viruses, the newly introduced HE 262 glycosylation site was retained and, instead, S1 A mutations that reduced S affinity were selected again, 263 with S-RBS Thr 83 replaced either by Ile (69%) -as seen before (Figs. 1A, 2C; Table 1 )-or by Asn (10%) 264 (Fig. 3D) . The latter mutation reduces S1 A affinity to 0.008 of that of wildtype.

Virus cloning by endpoint dilution yielded, in three out of five isolates, S1 A -Thr 83 Asn variants with the 266 newly introduced N-glycosylation site in HE intact (HE-Thr 114 Asn). Furthermore, a single S1 A -Thr 83 Ile 267 variant was isolated, but this virus in addition had the N-glycosylation site in HE destroyed (HE-

Thr 114 Asn/Ser 116 Phe) (Fig. 3D) . The observations led us to entertain the possibility that the mutations 269 in S1 A and HE did not occur independently and that, even in viruses expressing low affinity spikes, 270 partially restorative mutations in HE would yet provide a selective advantage. To test this, the clonal 271 S1 A -Thr 83 Asn/HE-Thr 114 Asn variants were serially passaged. All three viruses independently lost HE 272 Asn 114 glycosylation over time and, saliently, through Ser 116 Phe substitution exclusively. Even more 273 remarkably, with HE-Ser 116 Phe mutants gaining dominance, variants emerged that had restored S 274 affinity to (near)wildtype through substitution of S1 A -Asn 83 either by Thr or by Ser (Fig. 3E) . 275 For one of the five clonal populations obtained by endpoint dilution, we unfortunately failed to 276 determine its genotype for technical reasons. From the NGS analysis of the p1 population, we deduced 277 that the starting mutant must have been a low affinity S1 A -Leu 89 Pro variant that, like the S1 A -278 Thr 83 Asn/Ile variants described above, quickly lost the HE-Asn 114 glycan through an HE-Ser 116 Phe 279 substitution. Oddly enough, the Leu 89 Pro substitution had not been detected by NGS in the pre-cloning 280 virus stock. Note, however, that this mutation had been selected before twice independently in trials 281 with rBCoV-HE-F 211 A ( Table 2) . Possibly, it arose spontaneously during end point dilution procedure.

Be that as it may, its in vitro evolution proved informative (Fig. 4A) . NGS analysis of a passage p1 283 population, resulting from 120-hr multistep propagation, showed that 100% of the viruses coded for HE-Thr 114 Asn/Ser 116 Phe in combination with S1 A -Pro 89 (53.3%), -Thr 89 (40.5%), or -Ser 89 (2%). Note that 285 the relationship between these variants and the course of evolution -from Leu 89 in the parental 286 recombinant virus to Pro and from Pro to Thr or Ser-is evident from the codon sequences 287 (CTA→CCA→T/ACA) and that the Thr 89 and Ser 89 substitutions restored S RBS affinity almost to that 288 of wildtype RBS (Fig. 4B) . All three variants -S1 A -Pro 89 , -Thr 89 and -Ser 89 , were readily cloned and 289 isolated by standard endpoint dilution, and propagated independently without a requirement for 290 exogenous RDE. p1 also contained a minor population of viruses with parental S1 A , presumably 291 regenerated from S1 A -Pro 89 , which as for the S1 A -Thr 89 and Ser 89 variants would have required only a 292 single nucleotide substitution (CCA→CTA). Apparently, with HE lectin function partially restored, 293 viruses that regained (near) wildtype S affinity had a selective advantage. At the end of passage p2, 294 S1 A -Pro 89 variants had dwindled to less than 1.5%, S1 A -Thr 89 had become dominant at 75% and viruses 295 with parental S1 A -Leu 89 had rapidly risen from 2.25% in p1 to 23% (Fig. 4A) . 296 In summary, the introduction of a glycosylation site in the HE lectin domain that reduced receptor- catalytic virion release. We posit that in addition to an optimal balance between receptor-binding and 395 receptor-destruction, the system strives towards optimal virion avidity (Fig. 4C) . 396 Under natural circumstances, the set-point of the S/HE balance would be tailored to conditions met 553 1 Percental occurrence of BCoV S1 A mutations and 2 their relative binding affinities as measured by 554 equilibrium endpoint solid phase binding assay with S1 A -Fc fusion proteins with that of parental BCoV 555 S1 A -Fc ('wildtype') set at 1.0. S1 A variants Thr 22 Ile and Asn 27 Tyr emerged only in experiment 2 (see sFig.

556 5), but their affinities relative to that of wildtype S1 A are shown for comparison. (NA, not applicable). 

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