Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread. Author summary The main focus of research into the COVID-19 pandemic is the SARS-CoV-2 Spike (S) protein, which is the viral protein responsible for binding the ACE2 receptor on the host cell. Prior to and after attachment, the S needs to be activated by cellular proteases (e.g. furin, TMPRSS2), triggering the virus entry into the target cell. The unique feature of SARS-CoV-2 S is the presence of a multibasic site, an amino acid motif recognised by furin protease, and whose cleavage activates the S protein. Intensive antiviral research has been focused on developing furin inhibitors against SARS-CoV-2 infection. Here we show that furin is not absolutely required for SARS-CoV-2 virus production, suggesting that current SARS-CoV-2 antiviral therapies based on furin-targeting drugs may not completely prevent viral infection. Apart from the virus entry process, the S protein plays a key role in triggering cell-cell fusion, potentially allowing the quick spread of the virus among neighbouring cells. We show that furin-mediated pre-activation of S protein in virus infected cells is not necessary for triggering cell-cell fusion, while the multibasic site and the concomitant presence of TMPRSS2 protease on host cell membrane plays an important role in orchestrating the formation of multinucleated cells. Our results also suggest a key role of TMPRSS2 protease in promoting S-mediated cell-cell fusion paving the way for utilising a cocktail of inhibitors to efficiently treat SARS-CoV-2 infections.

Automated summary

Research suggests that furin plays a promoting role in SARS-CoV-2 infectivity and cell-cell spread, but it is not essential, indicating that furin inhibitors may reduce but not completely prevent viral spread.