Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

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.ImportanceAs 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.Article SummaryA 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.

SARS-CoV-2: Structure, Biology, and Structure-Based Therapeutics Development

“…The mutant virus also had reduced replication in Calu3 human respiratory cells and had attenuated disease in a hamster pathogenesis model. These results showed an important role of the furin cleavage site in SARS-CoV-2 replication and pathogenesis (Johnson et al, 2020).…”

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

“…Furthermore, the original 2003 SARS-CoV lacked a furin CS and was clearly transmitted between people, although this usually occurred during a symptomatic phase rather than asymptomatically or pre-symptomatically, thus allowing the virus outbreak to be controlled by public health measures (Liu, Gayle, Wilder-Smith, & Rocklov, 2020). Several recent studies have investigated the pathogenicity of naturally occurring or engineered deletion mutants in the hamster model, and shown that viruses lacking the furin CS are attenuated for replication and pathogenicity (Johnson et al, 2020; Lau et al, 2020; Wang et al, 2020; Wong et al, 2020). We, like others, did not find that SARS-CoV-2 induced significant clinical signs in ferrets, so were unable to assess the effect of the CS mutation on viral pathogenesis here.…”

Spike Glycoprotein-Mediated Entry of SARS Coronaviruses

“…Proteases in the extracellular milieu, such as trypsin, thermolysin, and furin, may enhance the preprocessing of the S protein and reduce the dependency of intracellular proteases [83]. Similarly, furin has been shown to cleave at the SARS-CoV-2 spike at the S1/S2 site [65,84]. These might all be hints of the adaptive evolution of coronaviruses.…”