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

Johnson, Bryan A.; Xie, Xuping; Bailey, Adam L.; Kalveram, Birte; Lokugamage, Kumari G.; Muruato, Antonio E.; Zou, Jing; Zhang, Xianwen; Juelich, Terry L.; Smith, Jennifer K.; Zhang, Lihong; Bopp, Nathen E.; Schindewolf, Craig; Vu, Michelle N; Vanderheiden, Abigail; Winkler, Emma S.; Swetnam, Daniele M.; Plante, Jessica A.; Aguilar, Patricia V.; Plante, Kenneth S.; Popov, Vsevolod L.; Lee, Benhur; Weaver, Scott C.; Suthar, Mehul S.; Routh, Andrew; Ren, Ping; Ku, Zhiqiang; An, Zhiqiang; Debbink, Kari; Diamond, Michael S.; Shi, Pei Yong; Freiberg, Alexander N.; Menachery, Vineet D.

doi:10.1038/s41586-021-03237-4

Cited by 667 publications

(731 citation statements)

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“…The furin-cleavage site in SARS-CoV-2 S is highly conserved in patient samples worldwide ( Figure 3D ), and is clearly important for virus pathogenesis [40]. However, a number of studies, including our own here, have reported that deletions in the furin-cleavage site can be acquired during passage in Vero cell substrates [32, 33].…”

Section: Resultsmentioning

confidence: 94%

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Pohl

Busnadiego

Kufner

et al. 2020

Preprint

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Since entering the human population, SARS-CoV-2 (the causative agent of COVID-19) has spread across the world, causing >40 million infections and >1 million deaths. While large-scale sequencing efforts have identified numerous genetic mutations in SARS-CoV-2 during its circulation, it remains largely unclear whether these changes impact adaptation, replication or transmission of the virus in its new host. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all the major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, viral stocks and passaging experiments, with kinetic virus replication data from non-human Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 sequence features that associate with distinct phenotypes. Notably, naturally-occurring substitutions in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G substitution generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E, and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin-cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human respiratory cells. Overall, our study uncovers natural sequence features in the SARS-CoV-2 genome that determine efficient virus replication and tropism for the human respiratory epithelium.

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Section: Resultsmentioning

confidence: 94%

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Pohl

Busnadiego

Kufner

et al. 2020

Preprint

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“…At least two emergent lineages of concern, B.1.1.7 (501Y.V1), and a newer variant whose prevalence is on the rise in Uganda both contain amino acid substitutions affecting the first position of the polybasic cleavage site, S:P681H and S:P681R, respectively (Andrew Rambaut, Nick Loman, Oliver Pybus, Wendy Barclay, Jeff Barrett, Alesandro Carabelli, Tom Connor, Tom Peacock, David L Robertson, Erik Volz, COVID-19 Genomics Consortium UK (CoG-UK), 2020; Lule Bugembe et al, 2021). The polybasic site strongly impacts viral replication in culture and as well as pathogenesis in animal models (Hoffmann, Kleine-Weber and Pöhlmann, 2020;Johnson et al, 2021). Although it is too early to predict whether any particular S: 677 polymorphic lineages will persist, given these observations, the recurrent parallelism affecting S: 677 suggests that this position will continue to surface in variants that show signs of increased transmissibility or fitness.…”

Section: Convergent Evolution Is a Hallmark Of Positive Selectionmentioning

confidence: 99%

Emergence in late 2020 of multiple lineages of SARS-CoV-2 Spike protein variants affecting amino acid position 677

Hodcroft

Domman

Snyder³

et al. 2021

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) plays critical roles in host cell entry. Non-synonymous substitutions affecting S are not uncommon and have become fixed in a number of SARS-CoV-2 lineages. A subset of such mutations enable escape from neutralizing antibodies or are thought to enhance transmission through mechanisms such as increased affinity for the cell entry receptor, angiotensin-converting enzyme 2 (ACE2). Independent genomic surveillance programs based in New Mexico and Louisiana contemporaneously detected the rapid rise of numerous clade 20G (lineage B.1.2) infections carrying a Q677P substitution in S. The variant was first detected in the US on October 23, yet between 01 Dec 2020 and 19 Jan 2021 it rose to represent 27.8% and 11.3% of all SARS-CoV-2 genomes sequenced from Louisiana and New Mexico, respectively. Q677P cases have been detected predominantly in the south central and southwest United States; as of 03 Feb 2021, GISAID data show 499 viral sequences of this variant from the USA. Phylogenetic analyses revealed the independent evolution and spread of at least six distinct Q677H sub-lineages, with first collection dates ranging from mid-August to late November 2020. Four 677H clades from clade 20G (B.1.2), 20A (B.1.234), and 20B (B.1.1.220, and B.1.1.222) each contain roughly 100 or fewer sequenced cases, while a distinct pair of clade 20G clusters are represented by 754 and 298 cases, respectively. Although sampling bias and founder effects may have contributed to the rise of S:677 polymorphic variants, the proximity of this position to the polybasic cleavage site at the S1/S2 boundary are consistent with its potential functional relevance during cell entry, suggesting parallel evolution of a trait that may confer an advantage in spread or transmission. Taken together, our findings demonstrate simultaneous convergent evolution, thus providing an impetus to further evaluate S:677 polymorphisms for effects on proteolytic processing, cell tropism, and transmissibility.

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“…We and others have previously shown that efficiency of S1/S2 cleavage in producer cells can modulate the entry efficiency of SARS-CoV-2 into different cell types (Hoffmann, Kleine-Weber, & Pöhlmann, 2020;Johnson et al, 2021;Peacock et al, 2020). Deleting the furin cleavage site enhances entry into cell lines that lack TMPRSS2 protease expression (e.g.…”

Section: Figure 2 -Comparative Replication Kinetics Of Sars-mentioning

confidence: 99%

“…P681H is at the S1/S2 cleavage site and could affect the efficiency of furin cleavage. We and others have previously shown that efficient cleavage of S at this site enhances transmissibility and pathogenicity of SARS-CoV-2 (Johnson et al, 2021;Peacock et al, 2020;Zhu et al, 2021). B.1.1.7 also harbours mutations of interest in other genes including a premature stop codon in ORF8, an accessory gene that likely enables immune evasion (Zhang et al, 2020), and a 3 amino acid deletion in NSP6, one of several proteins associated with virus regulation of the innate immune response (Xia et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

Brown

Goldhill

Zhou

et al. 2021

Preprint

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Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

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

Cited by 667 publications

References 45 publications

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Emergence in late 2020 of multiple lineages of SARS-CoV-2 Spike protein variants affecting amino acid position 677

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

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