Abstract
Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be considered. Results: Here we show that the genomes from SARS-CoV-2 and from SARS-CoV-1 are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for host cell invasion, virus replication, and host immune response. For SARS-CoV-1, we identify two sites: one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain; for SARS-CoV-2, one at the start of the viral replicase domain, and the other toward the end of the spike gene past its critical domain junction. At this junction, we detect a highly specific stretch of yeast DNA encoding for the critical furin cleavage site insert PRRA, which has not been seen in other lineage b betacoronaviruses. As yeast is not a natural host for this virus family, we propose a passage model for viral constructs in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Conclusions: These results provide evidence that among SARS-like coronaviruses only the genomes of SARS-CoV-1 and SARS-CoV-2 contain information that points to a synthetic passage in genetically modified yeast cells. Our data specifically allow the identification of the yeast S. cerevisiae as a potential recombination donor for the critical furin cleavage site in SARS-CoV-2.
Highlights
Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID19 pandemic
To obtain a genome-wide view of this homology signal we stacked together all homologous regions weighted by their individual alignment scores S, which resulted in an accumulated homology profile, Profiled alignment scores (pS)
While the peaks P1 and P2, as well as P4 and P5, could be positively cross-validated, the P3 signal in RaTG13 detected by BLASTlike Alignment Tool (BLAT) did not yield a statistically significant alignment with LALIGN, with its E-value reaching above 0.01. These highly differential data show that, for SARS-CoV-1 and for SARS-CoV-2, genes known to be critical for viral replication and host cell invasion display localized yeast homology at their flanking regions with limited extensions into the corresponding open reading frames. To explain this yeast DNA enrichment pattern, we propose the following artificial passage model (Figure 3A): Its starting point is a doubly auxotrophic, synthetic yeast cell line with stable, heterologous expression of viral replicase complex (RdRp, optionally together with auxiliary factors for replication, Aux) from a plasmid under the control of a selectable marker YSM1
Summary
Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID19 pandemic. For SARS-CoV-1, we identify two sites: one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain; for SARS-CoV2, one at the start of the viral replicase domain, and the other toward the end of the spike gene past its critical domain junction. At this junction, we detect a highly specific stretch of yeast DNA encoding for the critical furin cleavage site insert PRRA, which has not been seen in other lineage b betacoronaviruses. Keywords SARS related coronavirus, SARS-CoV-2, SARS-CoV-1, COVID-19, virus passage, yeast S. cerevisiae, directed evolution, genomic transformation, genome editing, synthetic biology
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