Cryo-EM structures of HKU2 and SADS-CoV spike glycoproteins and insights into coronavirus evolution

Jinfang Yu; Shuyuan Qiao; Runyu Guo; Xinquan Wang

However, the molecular mechanisms underlying the transmission of SADS-CoV from 68 bats to pigs are still unknown and need to be further explored. Recently it was shown 69 that SADS-CoV is able to infect cells from a broad range of species including mouse, 70 chicken, pig, monkey and human, indicating a high potential of the SADS-CoV for 71 interspecies transmission 17 . 72 The spike glycoprotein of coronaviruses mediates viral entry by binding host 73 receptor with the S1 subunit and fusing viral and cellular membranes with the S2 74 subunit, thereby determining viral host range and tissue tropism 18, 19 . As a class I viral 75 fusion protein, the spike exists on the envelope of virion as a homotrimer and each 76 monomer contains more than 1000 amino acid residues that can be cleaved into S1 and 77 S2 subunits 18 . For most coronaviruses, the N-terminal domain (NTD) of the S1 subunit HCoV-OC43 utilizes NTD to recognize glycans 29 ; and one exception is MHV, which 83 utilizes the NTD to bind mouse receptor CEACAM1a 30 . Therefore, the S1 subunit, 84 especially its NTD and CTD, is the most variable region of the spike, and is responsible 85 for different tropisms of coronaviruses. In comparison, the S2 subunit containing the 86 fusion peptide (FP) and heptad repeats (HR1 and HR2) for membrane fusion are more 87 conserved in both sequence and structure 18, 19 . For the SADS-CoV, receptor analysis 88 indicated that none of the known coronavirus protein receptors including ACE2, DPP4 89 and APN are essential for the cell entry 7,17 . There are also no reports regarding to the The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.02.23.961912 doi: bioRxiv preprint CoV 21,36-38 and MERS-CoV 36,39,40 ; the γ-coronavirus spike structure is determined for 98 IBV 41 and the δ-coronavirus spike structure is determined for PdCoV 42,43 . The cryo-99 EM structures of bat coronavirus spike trimers have not been reported, and only crystal 100 structures of the CTD from HKU4 44 , HKU5 45 and HKU9 46 were determined. 101 The spikes of SADS-CoV (1130 amino acid residues) and HKU2 (1128 amino 102 acid residues) are the shortest among all known coronavirus spike glycoproteins and 103 their amino acid identities to other known coronavirus spikes are lower than 28%, 104 indicating the spikes of HKU2 and SADS-CoV are unique [5] [6] [7] [8] [9] [10] 47 . In this study, we report 105 the cryo-EM structures of the SADS-CoV and HKU2 spike trimers at 2.83 Å and 2.38 106 Å resolution, respectively. The HKU2 spike trimer structure is the first one from bat 107 coronavirus. We analyzed the HKU2 and SADS-CoV trimer structures and also 108 compared the NTD, CTD, SD1 and SD2 domains of the S1 subunit and the S2 subunit