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

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.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

“…All three enzymes were shown to cleave the S1/S2 sequence in an enzymatic assay [41]. Also, the possibility that other proteases besides furin can process the S1/S2 site, is supported the observation that furin knockout does not fully prevent cleavage of the S1/S2 site in SARS-2-S [42]. When unprimed, SARS-2-S pseudoviruses are strongly boosted towards the cathepsin B/L route.…”

“…The multibasic motif is assumedly processed by furin-like proteases [37][38][39][40], but also other proteases have been proposed [41]. S1/S2 priming proved crucial for efficient SARS-2-S activation by TMPRSS2 and for viral entry in the airway epithelium Calu-3 cell model [37,38,42]. A minimal furin recognition motif is also present in the S protein of MERS-CoV (MERS-S) [43,44], but not the spike protein of SARS-CoV (SARS-S) [38].…”

A tale of three SARS-CoV-2 variants with independently acquired P681H mutations in New York State

“…In contrast to the recent decline in B.1.243 variants, other variants with a P681H mutation carrying T478K or S494P mutations have modestly increased in frequency in New York. P681 is located five residues upstream of a multibasic site in the Spike protein, the cleavage of which facilitates processes (such as membrane fusion) required for viral entry into the cell (Shang et al 2020; Papa et al 2021). This mutation also occurs in B.1.1.7 and could contribute to its increased rate of transmission (Davies et al 2021).…”

The evolutionary making of SARS-CoV-2

“…However, upon furin cleavage the D614G variant of spike has been reported to adopt preferentially a conformation where all three RBDs point down, with the consequence that their RBMs are masked and inaccessible for antibodies, but also for ACE2 (Gobeil, et al 2021). It is tempting to speculate about the role of secreted furin (Vidricaire, et al 1993) that may cleave spike even when it is not receptor bound, and of other proprotein convertases able to cleave at the multibasic furin site (Papa, et al 2021). It will be interesting to see if and how this apparently reversed role of furin cleavage on conformational masking in the D614G variant affects the virus’ strategy to evade the host immune system.…”

“…The functional importance of the furin cleavage extends beyond conformational masking. Pre-cleaved spikes allow the virus to infect cell types with low expression of TMPRSS2 (Shang, et al 2020a), and promote syncytia formation facilitating cell-cell transfer of the virus (Papa, et al 2021). However, depending on the cell type the presence of the furin cleavage site can be also strongly counter-selected.…”