The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin
Dear Editor,
The rapid spread of SARS-CoV-2 (also known as 2019-nCoV and HCoV-191), a novel lineage B betacoronavirus (βCoV), has caused a global pandemic of coronavirus disease (COVID-19). It has been speculated that RRAR, a unique furin-like cleavage site (FCS) in the spike protein (S), which is absent in other lineage B βCoVs, such as SARS-CoV, is responsible for its high infectivity and transmissibility.2
A coronavirus (CoV) infects the target cell by either cytoplasmic or endosomal membrane fusion. No matter what path it chooses, the final step of viral entry involves the release of RNA into the cytoplasm for replication. Therefore, the fusion capacity of the CoV-S is a leading indicator of infectivity of the corresponding virus. Consisting of S1 receptor-binding subunit and S2 fusion subunit, CoV-S needs to be primed through cleavage at S1/S2 site and S2′ site in order to mediate the membrane fusion (Fig. 1a). Previous studies have shown that an insertion of FCS consisting of multiple basic amino acids in the cleavage site of the haemagglutinin (HA) is associated with high virulence of influenza viruses.3 Coincidentally, phylogenetic analysis of SARS-CoV-2 identified an insertion of RRAR (FCS) at the S1/S2 site of SARS-CoV-2-S, which is absent in SARS-CoV and other SARS-related coronaviruses (SARSr-CoVs), particularly RaTG13, which has 96% identity of its genomic sequence to that of SARS-CoV-2 (Fig. 1b and Supplementary Fig. S1). Therefore, it has been speculated that this unique FCS may provide a gain-of-function, making SARS-CoV-2 easily enter into the host cell for infection, thus efficiently spreading throughout the human population, compared to other lineage B betacoronaviruses.2
Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis
2020 Aug 26.
Abstract
SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays.
Article Summary:
A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.
Importance
As COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis.
