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

Abstract

Porcine coronavirus SADS-CoV has been identified from suckling piglets with severe diarrhea in southern China in 2017. The SADS-CoV genome shares ~95% identity to that of bat α-coronavirus HKU2, suggesting that SADS-CoV may have emerged from a natural reservoir in bats. Here we report the cryo-EM structures of HKU2 and SADS-CoV spike (S) glycoprotein trimers at 2.38 Å and 2.83 Å resolution, respectively. We systematically compare the domains of HKU2 spike with those of α-, β-, γ-, and δ-coronavirus spikes, showing that the S1 subunit N- and C-terminal domains of HKU2/SADS-CoV are ancestral domains in the evolution of coronavirus spike proteins. The connecting region after the fusion peptide in the S2 subunit of HKU2/SADS-CoV adopts a unique conformation. These results structurally demonstrate a close evolutionary relationship between HKU2/SADS-CoV and β-coronavirus spikes and provide insights into the evolution and cross-species transmission of coronaviruses.

Fig. 1. Overall structures of HKU2 and SADS-CoV spike glycoproteins.

a Overall structures of HKU2 and SADS-CoV spike glycoproteins shown in side view (upper panel) and top view (lower panel). Three monomers of HKU2 spike are colored magenta, green, and orange, respectively; three monomers of SADS-CoV spike are colored pink, yellow, and cyan, respectively. The cryo-EM maps are shown as semitransparent surface and contoured at 2.6 RMS and 3 RMS for HKU2 and SADS-CoV spikes, respectively. The trigonal axes are shown as black dashed lines. Visible segments of each monomer are labeled accordingly. The cap, stalk, and root parts are partitioned by gray dashed lines. b Segmentation of HKU2 monomer. The segments of HKU2 are shown as boxes with the width related to the length of amino acid sequence. The start and end amino acids of each segment are labeled. The position of S1/S2, and S2′ cleavage sites are indicated. NTD N-terminal domain, CTD C-terminal domain, SD1 subdomain 1, SD2 subdomain 2, UH upstream helix, FP fusion peptide, CR connecting region, HR1 heptad repeat 1, CH central helix, BH β-hairpin, SD3 subdomain 3. c Overall structure of HKU2 monomer. Side views of HKU2 monomer shown in three directions. The segments are colored the same as in b.

Fig. 2. Structure of HKU2 NTD and comparisons.

a Structure of HKU2 NTD. Side views of HKU2 NTD are shown in two orthogonal directions. Disulfide bonds are shown as red sticks. Conserved disulfide bonds are labeled blue; other disulfide bonds are labeled black. Top sheet, middle sheet, bottom sheet, and bottom helix are partitioned by gray dashed lines. Comparison of HKU2 NTD and galectin3 are shown in the right panel. HKU2 NTD is colored salmon; galectin3 is colored green. PDB code: galectin3, 1A3K. b Subtype I NTD of α-coronavirus. Structural alignments of HKU2 NTD with HCoV-NL63 NTD1, and with HCoV-NL63 NTD2 are shown. HKU2 NTD is colored salmon. HCoV-NL63 NTD1 and NTD2 are colored gray. Disulfide bonds are shown as red sticks. Disulfide bonds conserved in both types of NTDs are indicated by red arrows; disulfide bonds conserved in subtype I NTD are indicated by blue arrows; PDB code: HCoV-NL63, 5SZS. c Subtype II NTD of α-coronavirus. Structural alignments of HCoV-229E NTD with HCoV-NL63 NTD1, and with HCoV-NL63 NTD2 are shown. HCoV-229E NTD is colored marine. HCoV-NL63 NTD1 and NTD2 are colored gray. Disulfide bonds conserved in both types of NTDs are indicated by red arrows; disulfide bonds conserved in subtype II NTD are indicated by orange arrows. PDB codes: HCoV-229E, 6U7H. d β-coronavirus NTDs resemble subtype I. Structural alignments of HKU2 NTD with SARS-CoV NTD, and with MHV NTD are shown. Disulfide bonds are shown and labeled the same as in b. Ceilings in β-coronavirus NTDs are shown in yellow. PDB codes: SARS, 5XLR; MHV, 3JCL. e γ-coronavirus IBV NTD resembles subtype I. Structural alignment of HKU2 NTD with IBV NTD is shown. Disulfide bonds are shown and labeled the same as in b. The additional loops in IBV NTD is shown in yellow. PDB code: IBV, 6CV0. f δ-coronavirus PdCoV NTD resembles subtype II. Structural alignment of HCoV-229E NTD with PdCoV NTD is shown. Disulfide bonds are shown and labeled the same as in c. The additional helices in PdCoV NTD are shown in yellow. PDB codes: HCoV-229E, 6U7H; PdCoV, 6B7N.

Fig. 3. Structure of HKU2 CTD and comparisons.

a HKU2 CTD shown in two opposite directions. Strands mentioned in the main text are labeled. The loop replaced by extra domain in β-coronavirus CTDs is shown in yellow. Disulfide bonds are shown as red sticks. Conserved disulfide bonds are labeled blue; other disulfide bonds are labeled black. b β-coronavirus CTDs belong to one-layer CTD. Structural alignments of HKU2 CTD with MERS-CoV CTD, and with MHV CTD are shown. Extra domains in β-coronavirus CTDs are colored yellow. Disulfide bonds are shown as red sticks. Disulfide bonds conserved in both one-layer CTD and two-layer CTD are indicated by red arrows. Disulfide bonds only conserved in one-layer CTD are indicated by blue arrows. PDB codes: MHV, 3JCL; MERS-CoV, 6Q05. c α-coronavirus (except for HKU2 and SADS-CoV), γ-coronavirus, and δ-coronavirus CTDs belong to two-layer CTD. Structural alignments of HCoV-NL63 CTD with PEDV CTD, with PdCoV CTD, and with IBV CTD are shown. Two layers of β-sheets are labeled. Extra loops in IBV CTD are colored yellow. Disulfide bonds are shown as red sticks. Disulfide bonds conserved in both one-layer CTD and two-layer CTD are indicated by red arrows. Disulfide bonds only conserved in two-layer CTD are indicated by orange arrows. PDB codes: HCoV-NL63, 5SZS; PEDV, 6U7K; IBV, 6CV0; PdCoV, 6B7N.

Fig. 4. Structures of SD1 and SD2 and comparisons.

a Comparison of SD1 from four genera of coronaviruses. HKU2 SD1 is colored salmon; SD1 from other coronaviruses are colored gray. Disulfide bonds are shown as red sticks. Red arrows indicate the disulfide bonds conserved in all genera of coronaviruses. PDB codes: HCoV-NL63, 5SZS; MHV, 3JCL; IBV, 6CV0; PdCoV, 6B7N. b Comparison of SD2 from four genera of coronaviruses. HKU2 SD2 is colored salmon; SD2 from other coronaviruses are colored gray. Disulfide bonds are shown as red sticks. Red arrows indicate the disulfide bonds conserved in all genera of coronaviruses. Blue arrows indicate the disulfide bonds only found in HKU2 (and SADS-CoV) and β-CoVs. The additional helices of SD2 from HKU2 (and SADS-CoV) and βCoVs are colored yellow and partitioned by gray dashed lines. PDB codes: HCoV-NL63, 5SZS; MHV, 3JCL; IBV, 6CV0; PdCoV, 6B7N.

Fig. 5. Quaternary packing of NTD and CTD.

a α-coronavirus S1 and δ-coronavirus S1 use intrasubunit packing pattern. NTD and CTD from the first monomer are colored blue, the second are colored red, and the third are colored magenta. PDB codes: HCoV-229E, 6U7H; PdCoV, 6B7N. b β-coronavirus S1 and γ-coronavirus S1 use cross-subunit packing pattern. NTD and CTD from the first monomer are colored blue, the second are colored red, and the third are colored magenta. The extra loop of IBV and the extra domains of β-CoVs are colored yellow and labeled as EX. PDB codes: MHV, 3JCL; IBV, 6CV0; SARS, 5XLR.

Fig. 6. Structure of HKU2 S2.

a Side views of HKU2 S2 shown in three directions. Seven segments of S2 are shown as different colors. UH upstream helix, FP fusion peptide, CR connecting region, HR1 heptad repeat 1, CH central helix, BH β-hairpin, SD3 subdomain 3. Disulfide bonds are shown as red sticks. Disulfide bonds conserved in all coronaviruses are labeled blue; the other disulfide bond is labeled black. b Conserved CR represented by MHV CR. CR and FP are colored the same as in a. Helices and strands in CR are labeled. R869 (S2′ cleavage site) is shown as stick and colored magenta. The blue dashed box shows R869 does not interact tightly with MHV CR. PDB code: MHV, 3JCL. c Unique feature of HKU2 CR. CR and FP are colored the same as in a. Helices and strands in CR are labeled. R671 (S2′ cleavage site) is shown as stick and colored magenta. The magenta dashed box shows detailed interactions between R671 and CR.