Abstract
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.
Highlights
In late 2019, a new coronavirus was identified in the Chinese province, Hubei, and named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for its close similarity to severe acute respiratory syndrome (SARS-CoV) that appeared in 2002
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
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
Summary
In late 2019, a new coronavirus was identified in the Chinese province, Hubei, and named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for its close similarity to severe acute respiratory syndrome (SARS-CoV) that appeared in 2002. S protein is a trimeric transmembrane glycoprotein whose ectodomain includes two main subunits: S1, responsible for attachment to the host cell receptor Angiotensin-Converting Enzyme 2 (ACE2) and for shielding the S2 subunit that contains the fusion machinery [4]. Similar to the fusion proteins of many other respiratory viruses, S protein of SARS-CoV-2 is activated by cellular protease-mediated cleavage [5,6,7,8]. Activation of S requires proteolytic cleavage at two distinct sites: in the unique multibasic site motif of Arg-Arg-Ala-Arg (RRAR), located between the S1 and S2 subunits, and within the S2 subunit (S2’) located immediately upstream of the hydrophobic fusion peptide that is responsible for triggering virus-cell membrane fusion [9,10,11]
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