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

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.