Coronavirus hemagglutinin-esterase and spike proteins co-evolve for functional balance and optimal virion avidity

Yifei Lang; Wentao Li; Zeshi Li; Danielle Koerhuis; Arthur C.S. van den Burg; Erik Rozemuller; Berend-Jan Bosch; Frank J.M. van Kuppeveld; Geert-Jan P.H. Boons; Eric G. Huizinga; Hilde M. van der Schaar; Raoul J. de Groot

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 . 225 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).