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Authors: Michael Letko et al. Link to paper: https://www.nature.com/articles/s41564-020-0688-y

Journal/ Pre-Print: Nature Microbiology (from bioRxiv)

Key Words: Host cell entry, viral receptor, ACE2, proteolytic processing

Research Highlights 

1. Receptor binding domain (RBD) of SARS-CoV-2 spike protein mediates entry into human cell lines via the receptor ACE2

2. Usage of ACE2 is highly specific and requires proteolytic pre-processing (by host proteases or exogenous proteases such as trypsin used in these experiments) for efficient cell entry

Summary 

The authors dissected the structural features of receptor usage and virus entry of lineage B β-coronaviruses, including SARS-CoV-2. To do this, the receptor-binding domain (RBD) of the SARS-CoV spike protein was replaced with other lineage B RBDs in pseudotyped VSV. They found that lineage B RBDs cluster into three functionally distinct clades. Clade 1 RBDs used ACE2 for cell entry, which was enhanced by S protein pre-processing by trypsin. ACE2-mediated entry required 14 ACE2-contacting amino acid residues found in clade 1 RBDs, plus regions immediately flanking them. Chimeric clade 2/3 spike proteins containing clade 1 RBD efficiently infected ACE2-expressing cells.

Impact for SARS-CoV2/COVID19 research efforts

Understand the virology and/or cell biology of SARS-CoV2/COVID19 - study provides structural evidence of the protein domains required for ACE2 usage by lineage B CoV, including SARS-CoV-2. Identified the critical RBD residues involved in ACE2-mediated host cell entry.

Study Type

· In vitro study - The authors generated pseudoviruses from 293T cells expressing constructs encoding SARS-CoV spike protein backbones containing RBDs from 29 different novel lineage B β-coronaviruses. These cells were then infected with empty VSV particles in order to produce VSV reporter particles pseudotyped with chimeric spike proteins. Virus entry into cell lines derived from different species was measured using a luciferase-based cell entry assay.

Strengths and limitations of the paper

Novelty: Identification of three different functional clades of lineage B β-coronaviruses based on receptor usage, which is determined by the unique structural features of the RBD that are related to physical interaction with ACE2.

Standing in the field: Confirms other recently published findings about SARS-CoV-2 using ACE2 for human cell entry.

Appropriate statistics: No statistical tests used. Technical replicates used instead of independent experiments.

Viral model used: As with other studies, authors used pseudotyped VSV particles containing SARS-CoV-2 RBD, with no confirmation using entire SARS-CoV-2 virus.

Translatability: None.

Main limitations: Do not use authentic SARS-CoV-2 virus, only the receptor binding domain.