ACE2 from Pipistrellus abramus bats is a receptor for HKU5 coronaviruses

Abstract

The merbecovirus subgenus of coronaviruses includes Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a zoonotic pathogen transmitted from dromedary camels to humans that causes severe respiratory disease. Viral discovery efforts uncover hundreds of merbecoviruses in different species across multiple continents, but few are studied under laboratory conditions, leaving basic questions regarding their human threat potential unresolved. Viral entry into host cells is a critical step for transmission between hosts. Here, we develop and apply a scalable approach to assesses novel merbecovirus cell entry across the entire merbecovirus subgenus. Merbecoviruses are sorted into clades based on the receptor-binding domain of the spike glycoprotein. Receptor tropism is clade-specific, with the clade including MERS-CoV using DPP4 and multiple clades using ACE2, including HKU5 bat coronaviruses. Mutational analysis identifies possible structural limitations to HKU5 adaptability and a cryo-EM structure of the HKU5-20s spike trimer reveals only 'down' RBDs.

Fig. 1. Identification of the functional RBD in merbecoviruses.

a The receptor binding domain in merbecoviruses engages the host receptor, DPP4 (PDB ID: 4L72, 5×59). Conserved glycines marking the functional RBD boundaries are indicated in yellow spheres. b Overview of MERS-CoV spike design for RBD replacement. c Chimeric spikes are used to generate single-cycle pseudotyped VSV reporter particles. d Overview of chimeric spike designs. e Amino acid sequence alignment of region flanking the RBD in merbecoviruses. f 293 T cell lines expressing indicated receptors were infected with pseudotypes and luciferase was measured for entry. Hashed bars represent chimeric spikes and solid bars represent full-length spikes as shown in panel (d). Cells were infected in quadruplicate as technical replicates. For each graph, individual replicates are plotted as points, the mean is shown as a bar and lines indicate standard deviation.

Fig. 2. Merbecovirus RBDs sort into clades based on sequence and entry phenotype.

a Total sequence diversity in merbecoviruses encompasses approximately 34 RBD sequences. b Cladogram showing the relationship of RBD amino acid sequences. Known receptors indicated. c Schematic overview of RBD clades, indicating the approximate size and location of indels characteristic to each merbecovirus RBD clade. d Western blot for FLAG (spike) expression of chimeric spikes. Results shown are representative of multiple biological replicates. e VeroE6 cells or Huh-7.5 cells were infected with VSV pseudotype panel bearing indicated chimeric spikes, in quadruplicate as technical replicates. The mean is shown as a bar and lines indicate standard deviation. Luciferase was measured for cell entry. f 293 T cells transduced to express human APN, human DPP4 or human ACE2 or transfected with Erinaceus europaeus ACE2, Erinaceus europaeus DPP4 or Pipistrellus abramus ACE2 were infected in the absence or presence of 100 μg/mL trypsin. g BHK-21 cells were transiently transfected with empty vector or Pipistrellus abramus ACE2, and infected with or without exogenous trypsin. All cells were infected in quadruplicate as technical replicates. Heatmaps represent mean relative entry as compared to pseudotypes without spike.

Fig. 3. Pipistrellus abramus ACE2 rescues entry of full-length HKU5 spike in non-susceptible cells.

a Pipistrellus abramus ACE2 expression cassette used in lentiviral transduction. b VeroE6 cells were transduced with Pipistrellus abramus ACE2 and infected with chimeric spike pseudotypes in quadruplicate as techincal replicates. The mean is shown as a bar and lines indicate standard deviation. c Schematic overview of full length and chimeric spikes. d Spike expression in cell lysates and incorporation in pseudotyped particles. e BHK-21 cells were transduced to express Pipistrellus abramus ACE2 and infected with pseudotypes bearing the indicated spikes. Solid circles represent full length spikes, hollow circles represent chimeric spikes. Cells were infected in quadruplicate as technical replicates. For each graph, individual replicates are plotted as points, the mean is shown as a bar and lines indicate standard deviation. f Overview of HKU5 molecular clone with GFP reporter in place of Orf5. g Light and fluorescent microscopy of indicated cell lines at 24-h post-infection. Scale bar in lower right corner is 400 μm. Data shown are representative of two biological replicates h Growth curve of viral replication as measured by PFU of supernatants at indicated time points. i Western blot of infected cells for viral and host proteins at 24-h post-infection.

Fig. 4. Recombinant HKU5 RBD and spike bind to Pipistrellus abramus ACE2.

Recombinant RBD or spike proteins were immobilized on ELISA plates and incubated with increasing amounts of the indicated receptor-Fc fusion protein or with a BSA-only control. Each data point is the mean of four measurements with bars indicating standard deviations. Binding to Pipstrellus abramus ACE2 is indicated in orange lines with circles, human ACE2 is indicated in red lines with squares, and human DPP4 is shown in blue with triangles.

Fig. 5. Clade consensus RBDs mimic clade-specific entry.

a Consensus sequences were generated for clades 1, 2, and 4. b 293 T cells transfected with indicated receptors were infected with chimeric spikes bearing clade consensus RBDs, in quadruplicate as technical replicates. The mean is shown as a bar and lines indicate standard deviation. c Schematic overview of sarbecovirus and merbecovirus RBD clades, indel patterns and receptor use.

Fig. 6. Structure-guided mutagenesis at the HKU5:ACE2 interface.

a Co-structure for PDF2180 RBD and Pipistrellus pipistrellus ACE2 (PDB ID: 7wpz). b AlphaFold3 predicted structures for HKU5-20s RBD (residues 390-614) and Pipistrellus abramus ACE2 (residues 20-608). c AlphaFold3 predicted structure for HKU5-21s RBD (residues 390-609) and Pipistrellus abramus ACE2 (residues 20-608). d Comparison of viral RBD footprints on species ACE2. e Mutant spike expression in cell lysates and incorporation in pseudotyped particles. ACE2 residues common to all three interfaces are highlighted. BHK or 293 T cells transduced with either f Pipistrellus abramus (P.a.) or g human (H.s.) ACE2 were infected with indicated pseudotypes in quadruplicate as technical replicates. Dark circles indicate wildtype sequences, and triangles indicate point mutant sequences. For each graph, four individual entry values are plotted as points, the mean is shown as a bar and lines indicate standard deviation. 2-way ANOVA with Tukey correction for multiple comparisons (***) Indicates P value < 0.001. h Entry values for mutant RBDs as compared to parental wild-type RBD.

Fig. 7. Structure of HKU5-20s spike trimer.

a EM density of HKU5-20s spike with three ‘down’ RBDs is shown from the side (left) and top (right). b Cartoon diagram of HKU5-20s spike trimer with different colors for each protomers shown from the side (left) and top (right). c Cartoon diagram of a spike protomer showing domains A-D. d Amino acid sequence conservation of 10 merbecovirus spike sequences calculated by ConSurf Database plotted on a surface representation of an HKU5-20s protomer (top) and HKU5-20s RBD (bottom). Yellow indicates that <10% of the sequences were available for the calculation at that position. e Cartoon diagram of the HKU5-20s RBD from two orientations.