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Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic¹-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses². To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

SUBMITTER: Johnson BA

PROVIDER: S-EPMC8175039 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Johnson Bryan A BA Xie Xuping X Bailey Adam L AL Kalveram Birte B Lokugamage Kumari G KG Muruato Antonio A Zou Jing J Zhang Xianwen X Juelich Terry T Smith Jennifer K JK Zhang Lihong L Bopp Nathen N Schindewolf Craig C Vu Michelle M Vanderheiden Abigail A Winkler Emma S ES Swetnam Daniele D Plante Jessica A JA Aguilar Patricia P Plante Kenneth S KS Popov Vsevolod V Lee Benhur B Weaver Scott C SC Suthar Mehul S MS Routh Andrew L AL Ren Ping P Ku Zhiqiang Z An Zhiqiang Z Debbink Kari K Diamond Michael S MS Shi Pei-Yong PY Freiberg Alexander N AN Menachery Vineet D VD

Nature 20210125 7849

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic<sup>1</sup>-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses<sup>2</sup>. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved ...[more]

PMID: 33494095

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Project description: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 (PRNT 50 ) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT 50 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 .

Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global coronavirus disease 2019 (COVID-19) pandemic, contains a unique, four amino acid (aa) "PRRA" insertion in the spike (S) protein that creates a transmembrane protease serine 2 (TMPRSS2)/furin cleavage site and enhances viral infectivity. More research into immunogenic epitopes and protective antibodies against this SARS-CoV-2 furin cleavage site is needed.MethodsCombining computational and experimental methods, we identified and characterized an immunogenic epitope overlapping the furin cleavage site that detects antibodies in COVID-19 patients and elicits strong antibody responses in immunized mice. We also identified a high-affinity monoclonal antibody from COVID-19 patient peripheral blood mononuclear cells; the antibody directly binds the furin cleavage site and protects against SARS-CoV-2 infection in a mouse model.FindingsThe presence of "PRRA" amino acids in the S protein of SARS-CoV-2 not only creates a furin cleavage site but also generates an immunogenic epitope that elicits an antibody response in COVID-19 patients. An antibody against this epitope protected against SARS-CoV-2 infection in mice.InterpretationThe immunogenic epitope and protective antibody we have identified may augment our strategy in handling COVID-19 epidemic.FundingThe National Natural Science Foundation of China (82102371, 91542201, 81925025, 82073181, and 81802870), the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (2021-I2M-1-047 and 2022-I2M-2-004), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2020-PT310-006, 2019XK310002, and 2018TX31001), the National Key Research and Development Project of China (2020YFC0841700), US National Institute of Health (NIH) funds grant AI158154, University of California Los Angeles (UCLA) AI and Charity Treks, and UCLA DGSOM BSCRC COVID-19 Award Program. H.Y. is supported by Natural Science Foundation of Jiangsu Province (BK20211554 andBE2022728).

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Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

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Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

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