A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein

Abstract

The unprecedented pandemic of pneumonia caused by a novel coronavirus, SARS-CoV-2, in China and beyond has had major public health impacts on a global scale [1, 2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomic analysis of samples from 227 bats collected from Yunnan Province in China between May and October 2019. Notably, RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity (61.3%) to SARS-CoV-2 in the receptor-binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong evidence that such insertion events can occur naturally in animal betacoronaviruses.

Keywords: COVID-19; S1/S2 cleavage site; SARS-CoV-2; bat coronavirus; spike protein.

Figure 1

Patterns of Sequence Identity between the Consensus Sequences of SARS-CoV-2 and Representative Beta-CoVs (A) Whole-genome similarity plot between SARS-CoV-2 and representative viruses listed in Table 1. The analysis was performed using Simplot, with a window size of 1,000 bp and a step size of 100 bp. (B) Similarity plot in the spike gene (positions 1–1,658) between SARS-CoV-2 and representative viruses listed in Table 1. The analysis was performed using Simplot, with a window size of 150 bp and a step size of 5 bp. See also Table S3.

Figure 2

Homology Modeling of the RBD Structures and Molecular Characterizations of the S1/S2 Cleavage Site of RmYN02 and Representative Beta-CoVs (A–D) Homology modeling and structural comparison of the RBD structures of RmYN02 and representative beta-CoVs, including (A) RmYN02, (B) RaTG13, (C) pangolin/MP789/2019, and (D) pangolin/GX/P5L/2017. The three-dimensional structures of the RBD from Bat-SL-CoV RmYN02, RaTG13, pangolin/MP789/2019, and pangolin/GX/P5L/2017 were modeled using the Swiss-Model program [13] employing the RBD of SARS-CoV (PDB: 2DD8) as a template. All the core subdomains are colored magenta, and the external subdomains of RmYN02, RaTG13, pangolin/MP789/2019, and pangolin/GX/P5L/2017 are colored cyan, green, orange, and yellow, respectively. The conserved disulfide bond in RaTG13, pangolin/GD, and pangolin/GX is highlighted, while it is missing in RmYN02 due to a sequence deletion. (E and F) Superimposition of the RBD structure of pangolin/MP789/2019 (E) and RmYN02 (F) with that of SARS-CoV-2. The two deletions located in respective loops in RmYN02 are highlighted using dotted cycles. (G) Molecular characterizations of the RBD of RmYN02 and the representative beta-CoVs. (H) Molecular characterizations of the cleavage site of RmYN02 and the representative beta-CoVs. See also Figures S2 and S3 and Table S2.

Figure 3

Phylogenetic Analysis of SARS-CoV-2 and Representative Viruses from the Subgenus Sarbecoronavirus (A) Phylogenetic tree of the full-length virus genome. (B) The S gene. (C) The RBD. (D) The RdRp. Phylogenetic analysis was performed using RAxML [21] with 1,000 bootstrap replicates, employing the GTR nucleotide substitution model. RBD is delimited as the gene region 991–1,572 of the spike gene according to [6]. All the trees are midpoint rooted for clarity. See also Figure S4.