Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients

Abstract

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site (PRRAR/S), generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We suggest that natural selection has favoured a "Don't burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.

Keywords: NGS; SARS-CoV-2; deletions; diversity; quasispecies; respiratory virus.

Figure 1.

Diagram showing location of the deletions found along the Spike gene and protein.

Figure 2.

Bar plot of deletions in amplicon N07 in the 18 patients (P01-P18) at the nucleotide level: Panel 1, patients with mild disease; Panel 2, patients with severe disease. The x axis provides the multiple alignment (MA) nucleotide positions and the amplitude of the deletions by subregions, and the y axis shows the frequency of the deletion (percentage) on the right and the number of reads on the left. As no insertions were observed, the MA positions correspond to S gene positions. Dashed lines indicates S1/S2 (left) and S2’ (right) cleavage sites. Bar plots for the 18 patients by amplicons are provided in supplementary material (Figures S1 to S14 for nucleotides and S15 to S27 for amino acids).

Figure 3.

Based on the life cycle of SARS-CoV, this diagram represents the hypothesis derived from our results. Entry of the virus in the host cell is shown at the top right of the diagram. At the transcription step, two scenarios are depicted: to the left, the viral particle resulting from normal S protein, and to the right the viral particle resulting from truncated S protein. In normal conditions, once the nucleoprotein is freed into the cytoplasm ss + RNA is translated into the non-structural proteins required for transcription. ss + RNA is transcribed into ss-RNA and later into genomic ss + RNA which is encapsidated (left side of the figure). Once the complete viral particle has been formed, it is secreted from the cell by exocytosis. The right side of the figure depicts the situation when a deletion occurs in the S gene during transcription of the complete genome and before subgenomic mRNAs are generated to produce the structural proteins. Translation of a deleted subgenomic spike mRNA would lead to a truncated S protein composed of the S1 domain without S2, which could be shed outside the cell as free S1. The box depicts possible destinations of free S1, which could bind to (1) the ACE2 cell receptor, (2) S1-specific neutralizing antibodies, or (3) free ACE2 receptor. ***The red triangle indicates the deletion in genomic RNA. ***Abbreviations: ACE2, angiotensin converting enzyme 2; mRNA, messenger RNA; NAb; neutralizing antibodies; pp1a, polyprotein 1a; RdRp, RNA-dependent RNA polymerase; S, spike; S1, subunit S1 at the N-terminal domain of the S protein, which includes receptor binding domain (RBD); S2, subunit S2 located at the C-terminal domain of S protein, which includes fusion peptide (FP), heptad repeat (HR) domain 1 and 2, and the transmembrane domain (TM); ss, single stranded; ss + RNA, single-stranded positive sense RNA; TMPRS22, human serine protease TMPRSS2.