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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Close relatives of MERS-CoV in bats use ACE2 as their functional receptors
Qing Xiong1, Lei Cao2, Chengbao Ma1
1State Key Laboratory of Virology, Institute for Vaccine Research and Modern Virology Research Center, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Wuhan University, Wuhan, China.
Abstract:
Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.
Insights
Middle East respiratory syndrome coronavirus (MERS-CoV)-related bat viruses, NeoCoV and PDF-2180, utilize specific bat angiotensin-converting enzyme 2 (ACE2) as entry receptors. This discovery highlights potential zoonotic threats and promiscuous receptor usage among coronaviruses.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Middle East respiratory syndrome coronavirus (MERS-CoV) and some bat coronaviruses use dipeptidyl peptidase-4 (DPP4) for cell entry.
- The entry receptor for NeoCoV, a bat MERS-CoV relative, was previously unknown.
Purpose of the Study:
- To identify the entry receptor for NeoCoV and its relative PDF-2180.
- To characterize the interaction between NeoCoV/PDF-2180 spike proteins and ACE2.
- To assess potential cross-neutralization with existing coronavirus antibodies.
Main Methods:
- Pseudotype virus entry assays were used to test viral binding and entry.
- Cryo-electron microscopy was employed to visualize the spike protein-receptor interface.
- Site-directed mutagenesis identified key residues in ACE2 and the spike protein RBD.
Main Results:
- NeoCoV and PDF-2180 efficiently use specific bat ACE2 orthologs and human ACE2 as entry receptors via their spike protein receptor-binding domains (RBDs).
- Cryo-EM revealed a distinct RBD-ACE2 binding interface involving protein-glycan interactions.
- Specific residues in human ACE2 restrict NeoCoV entry, while an RBD mutation facilitates entry into human cells. Broadly neutralizing antibodies targeting ACE2 or betacoronaviruses inhibited NeoCoV/PDF-2180.
Conclusions:
- MERS-CoV-related viruses can utilize ACE2 as an entry receptor, demonstrating receptor promiscuity.
- The findings reveal a potential zoonotic threat posed by these bat coronaviruses.
- Understanding these interactions is crucial for developing broad-spectrum antiviral strategies against emerging coronaviruses.
