Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a genome comprised of a ~30K nucleotides non-segmented, positive single-stranded RNA. Although its RNA-dependent RNA polymerase exhibits exonuclease proofreading activity, viral sequence diversity can be induced by replication errors and host factors. These variations can be observed in the population of viral sequences isolated from infected host cells and are not necessarily reflected in the genome of transmitted founder viruses. We profiled intra-sample genetic diversity of SARS-CoV-2 variants using 15,289 high-throughput sequencing datasets from infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing, and/or sequencing protocols. Despite high mutational background, we confidently identified intra-variable positions recurrent in the samples analyzed, including several positions at the end of the gene encoding the viral S protein. Notably, most of the samples possesses a C->A missense mutation resulting in the S protein lacking the last 20 amino acids (SΔ20). Here we demonstrate that SΔ20 exhibits increased cell-to-cell fusion and syncytia formations. Our findings are suggestive of the consistent emergence of high-frequency viral quasispecies that are not horizontally transmitted but involved in intra-host infection and spread.Author summaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease, COVID-19, has caused significant worldwide mortality and unprecedented economic burden. Here we studied the intra-host genetic diversity of SARS-CoV-2 genomes and identified a high-frequency and recurrent non-sense mutation yielding a truncated form of the viral spike protein, in both human COVID-19 samples and in cell culture experiments. Through the use of a functional assay, we observed that this truncated spike protein displays an elevated fusogenic potential and forms syncytia. Given the high frequency at which this mutation independently arises across various samples, it can be hypothesized that this deletion mutation provides a selective advantage to viral replication and may also have a role in pathogenesis in humans.

Meta-Analysis and Structural Dynamics of the Emergence of Genetic Variants of SARS-CoV-2

“…Another caveat in analyzing sequences in the GISAID database is that consensus sequences are uploaded, but subsequences and quasispecies are generally not included. Therefore, it is possible that mutations in sub-variants may be missed that could contribute to virus replication, pathogenicity, and spread ( Rocheleau et al, 2021 ). A global approach to analyzing both transmitted variants and non-transmitted sub-variants and quasispecies could provide a better understanding of the effects of SARS-CoV-2 mutations.…”

Meta-Analysis and Structural Dynamics of the Emergence of Genetic Variants of SARS-CoV-2

“…Another caveat in analyzing sequences in the GISAID database is that consensus sequences are uploaded, but subsequences and quasispecies are generally not included. Therefore, it is possible that mutations in sub-variants may be missed that could contribute to virus replication, pathogenicity, and spread (52). A global approach to analyzing both transmitted variants and non-transmitted sub-variants and quasispecies could provide a better understanding of the effects of SARS-CoV-2 mutations.…”

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen

“…To more conclusively determine bifonazole's effect on VSV-Spike viral entry, we interrogated its effect on fusion between spike and ACE2 using a bimolecular fluorescence complementation (BiFC) assay. 18 Briefly, HEK293T cells expressing human ACE2 (hACE2), hACE2/TMPRSS2, or pCaggs, and Zip-Venus-1 (target cells) were seeded in dishes overnight and then pre-treated with 0, 25, 50, or 100 mM bifonazole. Next, HEK293T cells expressing spike and Zip-Venus-2 (effector cells) were added to the wells.…”