henhaobio
Sign in

CellBiochemistryPeer-reviewed journal article

Molecular basis of convergent evolution of ACE2 receptor utilization among HKU5 coronaviruses

Young‐Jun ParkiD, Chen LiuiD, Jimin LeeiD, Jack T BrowniD, Chengbao Ma, Peng LiuiD, Risako GeniD, Qing XiongiD, Samantha K. ZepedaiD, Cameron StewartiD, Amin AddetiaiD, Caroline J. CraigiD, M. Alejandra TortoriciiD, Abeer N. AlshukairiiD, Tyler N. StarriD, Huan YaniD, David VeesleriD

PDFDOIPreprintCodeData
In-depth full-text digest available · Jump to digest ↓

Abstract

DPP4 was considered a canonical receptor for merbecoviruses until the recent discovery of African bat-borne MERS-related coronaviruses using ACE2. The extent and diversity with which merbecoviruses engage ACE2 and their receptor species tropism remain unknown. Here, we reveal that HKU5 enters host cells utilizing Pipistrellus abramus (P.abr) and several non-bat mammalian ACE2s through a binding mode distinct from that of any other known ACE2-using coronaviruses. These results show that several merbecovirus clades independently evolved ACE2 utilization, which appears to be a broadly shared property among these pathogens, through an extraordinary diversity of ACE2 recognition modes. We show that MERS-CoV and HKU5 have markedly distinct antigenicity, due to extensive genetic divergence, and identified several HKU5 inhibitors, including two clinical compounds. Our findings profoundly alter our understanding of coronavirus evolution and pave the way for developing countermeasures against viruses poised for human emergence.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (28 Aug 2024) · CC BY 4.0

HKU5 merbecoviruses use bat Pipistrellus abramus ACE2 and several artiodactyl ACE2s through a cryo-EM-defined binding mode distinct from other ACE2-using coronaviruses, showing independent evolution of ACE2 usage.

DPP4 was long assumed to be the universal merbecovirus receptor, and how widely merbecoviruses use ACE2, and in which host species, was unknown. This work maps HKU5 ACE2 tropism across 117 orthologs, resolves the interface structurally, and tests candidate countermeasures.

Key results

  1. ShownHKU5-19s RBD bound only P.abr ACE2 among the bat orthologs tested, plus several non-bat mammalian ACE2s (Artiodactyl and Carnivore species); pseudovirus entry confirmed these and also occurred with R.nor ACE2.

    RBD-hFc screen of 117 ACE2 orthologs (62 bat); non-bat binders included B.tau and M.erm ACE2. R.nor allowed pseudovirus entry without detectable RBD binding (attributed to avidity and trypsin). Caco-2/P.abr ACE2 supported psVSV-HKU5-19s and authentic HKU5-1 propagation. · HKU5 utilizes ACE2 as entry receptor; Figure legends

  2. ShownCryo-EM resolves the HKU5-19s RBD on the P.abr ACE2 peptidase domain. HKU5 engages ACE2 with the RBM β-sheet face that MERS-CoV and HKU4 use for DPP4, whereas NeoCoV uses the RBM tip. P.abr ACE2 binds HKU5-19s with higher affinity than HKU5-33s.

    3.1 Å reconstruction from 550,684 particles (EMD-46512, PDB 9D32); ∼950 Å buried surface per partner; ACE2 99% monomeric in selected 2D classes; BLI showed ∼2-fold higher P.abr ACE2 affinity for HKU5-19s vs HKU5-33s, driven by slower off-rates. · Molecular basis of HKU5 recognition of ACE2; Cryo-electron microscopy data collection…; Data and code availability

  3. ShownMutagenesis defines four ACE2 tropism determinants (P.abr positions 321, 324, 328-329, 353). Placing R/D at human ACE2 329/330 plus a third substitution (N322Y, Q325P or G354R) gives robust pseudovirus entry.

    BLI: HKU5-19s Y464A, Y558A, A517R, G519W abolished P.abr ACE2 binding; Y518A nearly did. Human ACE2 E329R/N330D gave weak entry; adding N322Y, Q325P or G354R gave robust entry. Pseudovirus assays n=3 biological replicates, unpaired t-tests. · Determinants of ACE2 species tropism; Figure legends; Statistical analysis

  4. ShownMERS-CoV infection-elicited plasma cross-neutralizes HKU5-19s pseudovirus only rarely and weakly, consistent with large antigenic distance; S2-directed broadly neutralizing antibodies still inhibit HKU5.

    At least 50% entry inhibition at 1/10 dilution for 3 of 28 plasma samples (15, 16, 24); MERS-CoV and HKU5 share ∼65% (S) and ∼50% (RBD) amino acid identity. S2P6, B6 and 76E1 inhibited authentic HKU5-1 in Caco-2/P.abr ACE2 cells. · Identification of HKU5 countermeasures; Discussion

  5. SuggestedProtease, fusion and polymerase inhibitors block authentic HKU5-1 propagation in P.abr ACE2-expressing Caco-2 cells, implicating TMPRSS2-type activation and endosomal entry not dependent on cysteine proteases.

    Camostat at 10 µM, bafilomycin A1 at 1 µM, EK1C4 at 1 µM and remdesevir at 10 µM inhibited propagation; E64d did not. Readout was anti-HKU5 N immunofluorescence at 24 hpi; no IC50 values reported. · Identification of HKU5 countermeasures; Figure legends

Methods, data and reproducibility

Receptor screening by RBD-Fc binding and VSV pseudovirus entry across 117 ACE2 orthologs, cryo-EM of the HKU5-19s RBD with P.abr ACE2, BLI and mutagenesis for determinants, then neutralization and inhibitor assays plus RBD phylogenetics.

  • Mammalian ACE2 orthologs screened for HKU5-19s RBD binding and pseudovirus entry (bat and non-bat) · Multiple mammals (bat and non-bat) · n = 117 orthologs (62 bat)
  • Cryo-EM complex of HKU5-19s RBD with P.abr ACE2 ectodomain · Pipistrellus abramus ACE2; HKU5 virus RBD · n = 11,223 micrographs; 550,684 particles in final map · EMD-46512; PDB 9D32
  • Merbecovirus RBD sequences for maximum-likelihood phylogeny · Merbecoviruses · n = 71 unique RBD sequences
  • Plasma from hospitalized MERS-CoV-infected individuals sampled before the COVID-19 pandemic, tested for HKU5-19s pseudovirus neutralization · human · n = 28 plasma samples
  • Authentic HKU5-1 propagation in Caco-2 cells with or without stable P.abr or human ACE2 · Human cell line (Caco-2); HKU5-1
Methods & tools
cryoSPARC, Warp, Topaz, RELION, Rosetta, AlphaFold3, MAFFT-DASH, GARD, RAxML (WAG+G), IQ-TREE2, Biolayer interferometry (Octet RED96), VSV pseudovirus entry assay
Software & environment
MEGA-X 10.1.8 (MUSCLE alignment), IQ-TREE2 with Q.plant+I+R3 and 1000 bootstraps, iTOL v6, GraphPad Prism 8 and 10; no containers stated.
Reproducibility gaps
Code is provided only for the phylogenetic pipeline; none stated for assay quantification or structure analysis. Plasma demographics are in Table 3 and cited from earlier work.
Limitations (4)
  • No human ACE2-mediated HKU5 RBD binding or pseudovirus entry was detected; authentic HKU5-1 replicated only weakly in human Caco-2 cells with exogenous trypsin. · stated by the authors
  • Pseudovirus entry with R.nor ACE2 occurred without detectable RBD binding, which the authors attribute likely to avidity from multivalent S trimers and the use of trypsin. · noted in this summary
  • Effects of K520G and Y545G may be modulated by epistasis in natural isolates, and sparse bat sampling limits knowledge of HKU5 diversity. · stated by the authors
  • The structure covers only HKU5-19s RBD with P.abr ACE2; other isolates are inferred from binding assays. Some pseudovirus panels are single experiments, and inhibitor data lack dose-response potency estimates. · noted in this summary

Read this if you study coronavirus receptor switching, ACE2 species tropism, or merbecovirus spillover risk and want structure-guided tropism determinants and candidate inhibitors.

Citations
47
FWCI
29.37
Field percentile
top 1%

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISLiu '25Alfajaro '25Catanzaro '24Ma '23Zhang '25Ma '22
89% semantic matchMicrobiology
Chen Liu, Young‐Jun Park, Chengbao Ma+10 more·bioRxiv·Feb 19, 2025· 6 citations