Functional assessment of cell entry and receptor usage for SARS-Cov-2 and other lineage B betacoronaviruses

Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020;5(4):562‐569. doi:10.1038/s41564-020-0688-y

Review written by:

Ariel Pourmorady, edited by Eunice Lee

Green - published in Nature Microbiology; straightforward molecular biology that directly relates RBD sequence and structure to function.

The receptor binding domain (RBD) of the coronavirus spike protein contains all sequence information necessary for proper folding and host receptor binding. In addition to SARS-CoV and SARS-CoV-2, there are approximately 200 lineage B betacoronaviruses with published sequences, which together represent 29 unique RBDs; however, their potential to infect humans remains unknown. Here, Letko et al. establish an experimental pipeline to screen these 29 RBDs for 1) ability to infect a variety of human and non-human cell types and 2) usage of specific known human receptors of coronaviruses.

A phylogenetic analysis of the 29 RBDs uncovered 3 clades. Letko et al. then replaced the RBD sequence of the SARS-CoV spike protein with each of the unique RBDs, and used VSV reporter particles to infect a variety of cell types, both human and non-human. Regardless of cell type, all VSV particles containing RBDs from Clade 1 (which includes SARS-CoV) were able to infect the target cell, while none of the RBDs in other clades were able to promote host cell entry.

Upon binding to the host receptor, a host cell protease cleaves the spike protein to allow for viral entry. The authors tested whether addition of an exogenous protease would enhance the entry of viral particles generated from each of the 29 RBDs. Only RBDs from Clade 1 were able to infect host cells without exogenous protease. A subset of RBDs from Clade 2 showed enhanced entry upon protease treatment. RBDs from Clade 3 were not affected by protease treatment.

They then tested human receptor specificity by infecting cells expressing known coronavirus receptors (ACE2, DPP4, APN) with spike chimeras generated from HCoV-229E, an alphacoronavirus, and a series of betacoronaviruses (including SARS-CoV, SARS-CoV-2, and MERS-CoV) with or without exogenous protease treatment. Only SARS-CoV-2 and Clade 1 betacoronavirus RBDs were able to infect cells expressing human ACE2, and did not enter cells expressing DPP4, APN, or no receptor. Human DPP4 and APN only mediated the entry of MERS-CoV and HCoV-229E, respectively.

Letko et al. also sought to identify the determinants of ACE2 usage by clade 1 lineage B betacoronaviruses. They focus on the 14 essential amino acid residues embedded within a receptor-binding motif (RBM) located at the C-terminal end of the RBD known to interact with human ACE2, and introduce various mutations in Clade 2 and Clade 3 chimeras to encourage ACE2 utilization. Only by replacing all 14 amino acids and the surrounding amino acid sequence of the RBM, including the loops, are Clade 2/3 chimeras able to infect ACE2+ cells. Finally, they generate full-length Clade 2 and Clade 3 spikes with a Clade 1 RBD. Whereas a full-length Clade 1 spike was capable of infecting an ACE2+ cell, independent of protease treatment, the newly generated chimeras depend on protease treatment for infectivity.

The results of this study reveal that other, previously unstudied, betacoronaviruses may be capable of infecting human cells in a protease-dependent or protease-independent fashion. The authors also suggest their screening method can be expanded to test zoonotic potential of other newly discovered viruses.

Review Notes

1. Study was entirely in vitro and therefore may not be representative of infectivity in vivo.

2. The panel of human receptors investigated were limited to 3 (ACE2, DPP4, APN). CoV might have affinity for human receptors not tested in this study.

3. The study was only performed on pseudotyped VSV. There may be other factors on the CoV viral surface that may facilitate different parts of viral entry.

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