PMC

Comparative sequence analysis of the nsp16-HE gene junction. Multiple alignments of the nucleotide sequences of CoV HKU1 genotypes A, B, and C. In CoV HKU1 genotype A and CoV HKU1 genotype B, only the nucleotides differing from those in CoV HKU1 genotype C are depicted. The nucleotides in CoV HKU1 genotype C that are the same as those in CoV HKU1 genotype A but different from those in CoV HKU1 genotype B are highlighted in gray, and those in CoV HKU1 genotype C that are the same as those in CoV HKU1 genotype B but different from those in CoV HKU1 genotype A are highlighted in black. The putative template switching region is underlined and bold. The stop codon of ORF1ab (TAG) and the start codon of the HE gene (ATG) are also underlined. The arrows denote positions with nucleotide polymorphisms (at 21297, N6, N7, N9, N10, N11, N13, N14, N23, and N24 [genotype A] were T instead of G; at 21429, N6 [genotype A] was C instead of T; at 21576, N15 [genotype B] was C instead of T; at 21908, N15 and N25 [genotype B] were G instead of A; and at 21949, N14 was T instead of C).

Comparative sequence analysis of the nsp6-nsp7 junction. Multiple alignment of the nucleotide sequences of CoV HKU1 genotypes A, B, and C. In CoV HKU1 genotype B and CoV HKU1 genotype C, only the nucleotides differing from those in CoV HKU1 genotype A are depicted. The nucleotides in CoV HKU1 genotype C that are the same as those in CoV HKU1 genotype A but different from those in CoV HKU1 genotype B are highlighted in black, and those in CoV HKU1 genotype B that are the same as those in CoV HKU1 genotype A but different from those in CoV HKU1 genotype C are highlighted in gray. The putative template switching region is underlined and bold. The first (TCA) and last (CAG) codons of nsp7 are also underlined. The arrows denote positions with nucleotide polymorphism (at position 11414, N16, N17, N20, N21, and N22 [genotype C] were T instead of C; at 11422, N16 and N20 [genotype C] were C instead of T; at 11449, N25 [genotype B] was C instead of T; at 11528, N16, N17, N20, and N22 [genotype C] were C instead of T; at 11740, N6, N7, N9, N10, N11, N13, N14, N18, N23, and N24 [genotype A] were T instead of C; at 12095, N6 and N7 [genotype A] were T instead of C; at 12140, N23 and N24 [genotype A] were C instead of T; at 12367, N15 and N25 [genotype C] were A instead of G; and at 12400, N6, N7, N9, N10, N11, N13, N14, N18, N23, and N24 [genotype A] were C instead of T).

HKU1 S1^A site B mediates binding to 9-O-Ac-Sias. (A) HKU1 S1^A–Fc binds to 9-O-Ac-Sia through low-affinity, high-specificity interactions. (Upper) Conventional HAA (top row) and high-sensitivity pA-LS NP-HAA (bottom rows) with native HKU1 S1^A-Fc (HKU1) or mutants thereof (Lys⁸⁰Ala, Thr⁸²Ala, Trp⁸⁹Ala). Noncomplexed nanoparticles were included as negative control (−). (Lower) NP-HAA with erythrocytes (mock)depleted for 9-O-Ac-Sias by prior sialate-O-acetylesterase treatment. (B) Surface representation of site B in BCoV (Top) and HKU1 (Middle) [PDB ID code 5I08 ()], respectively. Protein structures were aligned with site B residues Lys⁸¹, Thr⁸³, and Trp⁹⁰ as query. Side chains of site B residues shown in sticks. BCoV residues 29–34 (element e1) and 246–253 (e2), and corresponding elements in HKU1 indicated in orange. (Bottom) Overlay of cartoon representations of BCoV (purple) and HKU1 (white) S1^A centered on site B. In the HKU1 protein, e1 and e2 are colored orange and marked. Side chains of Asn²⁹ and Asn²⁵¹, acceptors of N-linked glycosylation, indicated in sticks. BCoV Trp⁹⁰ and its HKU1 ortholog, also in sticks, shown as a reference point to site B. (C, Upper) Replacement of HKU1 S1^A e1 and e2 by corresponding BCoV elements increases binding affinity to 9-O-Ac-sialoglycans present on rat erythrocytes. NP-HAA with native HKU1 S1^A–Fc (HKU1) or with HKU1 S1^A–Fc derivatives, with e1 and e2 replaced by the corresponding BCoV elements separately (B-e1, B-e2) or in combination (B-e1+e2). For comparison, mutant HKU1 S1^A–Fc proteins were included with N-glycosylation sites eliminated in site A (N¹⁷¹Q) or in elements e1 (N²⁹Q) and e2 (N²⁵¹Q), individually and in combination (N²⁹Q + N²⁵¹Q). (C, Lower) NP-HAA with erythrocytes (mock)depleted for 9-O-Ac-Sias. (D) Replacement of HKU1 S1^A e1 and e2 by corresponding BCoV elements increases binding affinity to 9-O-sialoglycans as to allow detection by conventional HAA. (E) Replacement of HKU1 S1^A e1 and e2 increases binding affinity as to allow detection of 9-O-Ac-sialoglycans by nanoparticle sp-LBA. (Left) Conventional sp-LBA with soluble S1^A-Fc. (Right) Nanoparticle sp-LBA with S1^A–Fc complexed to pA-LS icosahedral shells in twofold serial dilutions, starting at 50 nmol pA-LS per well. Data points are mean averages of independent duplicate experiments, each performed in triplicate.

a Schematic diagrams of HKU1 spike S1-RBD and TMPRSS2 constructs. b, c BLI titrations of 1A RBD–TMPRSS2 (b) and 1B RBD–TMPRSS2 (c); the K_D values of binding are indicated. Data fitting is shown with red curves. d Cryo-EM structure of HKU1 1A RBD (orange) in complex with TMPRSS2 (purple); left, cryo-EM densities, right, ribbon models. e Cryo-EM structure of HKU1 1B RBD (magenta) in complex with TMPRSS2 (blue); left, cryo-EM densities, right, ribbon models. Sugars are colored green. f 1B RBD and TMPRSS2 interaction shown with ribbon models. HKU1 receptor binding groove (RBG) is indicated. g Sequence alignment of 1A-RBD and 1B-RBD illustrating 13 residues (marked by blue asterisks) of RBG important for TMPRSS2 binding. Two disulfide bridges stabilizing RBG are indicated. h, i Detailed interaction between HKU1 1B RBD and TMPRSS2. TMPRSS2 is shown with molecular surface (blue), the proteinase active site is colored red, and RBD binding site is colored yellow. HKU1 1B RBD is shown with ribbon model (magenta). Key interaction residues are shown with stick model. Magnified view focusing 1B-RBD β-hairpin–TMPRSS2 interaction (h) and 1B-RBD Proline-rich–TMPRSS2 interaction (i) are shown. j HKU1 RBD–TMPRSS2 complex. RBD core domain is colored orange, RBG is colored red, and the insertion domain (RBM) is colored magenta. The black box indicates the RBD–TMPRSS2 interface. k, l TMPRSS2 (blue) is modeled to an HKU1 spike trimer with one RBD in up-state. Ribbon model (k) and molecular surface (l) are shown. Sugars are shown with green spheres; the RBGs are colored red. m Top view of an HKU1 spike trimer in close-state. RBGs are colored red. n A working model of HKU1 spike protein-mediated receptor binding.