Virological characterization and biological significance of the spike furin cleavage site in the Omicron variant of SARS-CoV-2 in vitro and in vivo

Abstract

<p><strong>Introduction </strong></p><p>Acquisition of a polybasic furin cleavage site (FCS) may provide SARS-CoV-2 with a unique ability to cross host barriers and facilitate human infections during the early stages of cross-species transmission. However, it is currently unclear whether the human adapted Omicron variant of SARS-CoV-2 requires FCS for efficient replication in mammalian cells.</p><p><strong>Methods</strong></p><p>We used a reverse genetic system to generate a series of mutant viruses containing complete polybasic FCS deletion or various FCS mutations. We infected human airway epithelium (HAE) air-liquid interface cell-culture models (ALI) for single-cell temporal transcriptomic analysis. Additionally, a newly developed spatial transcriptomics technology was used to investigate tissue tropism of virus with variations in FCS and in situ virus-host interaction.</p><p><strong>Results</strong></p><p>Our study found that the wildtype (WT) SARS-CoV-2 virus (Omicron BA.5) replicated effectively in the HAE ALI model and mainly targeted the ciliated cells. However, the Del-FCS Omicron virus substantially attenuated the replication efficiency in the ALI system compared to the WT virus. Notably, WT virus infection damaged ciliated cells more significantly than the Del-FCS virus, leading to the loss of their motile cilia, but this was not observed in Del-FCS infection. Single-cell RNAseq analysis of infection with different FCS mutated viruses and WT virus in the ALI system showed that FCS is required for efficient replication of SARS-CoV-2 Omicron variant, even after it is fully adapted to humans. We also used a pangolin SARS-CoV-2 like virus for comparison in this study.</p><p><strong>Conclusions</strong></p><p>Our study demonstrated that HAE ALI is a suitable model for studying molecular basis of host adaption by SARS-CoV-2 coronavirus to address the importance of polybasic FCS during infection. The FCS is required for cross-species transmission of SARS-CoV-2 virus at the early stage of human infections and is stably retained for efficient replication in the most recent Omicron variant.</p>