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

Peacock, Thomas P.; Goldhill, Daniel H.; Zhou, Jie; Baillon, Laury; Frise, Rebecca; Swann, Olivia C.; Kugathasan, Ruthiran; Penn, Rebecca; Brown, Jonathan C.; Sanchez-David, Raul Y.; Braga, Luca; Williamson, Maia Kavanagh; Hassard, Jack A.; Staller, Ecco; Hanley, Brian; Osborn, Michael; Giacca, Mauro; Davidson, Andrew D.; Matthews, David A.; Barclay, William

doi:10.1101/2020.09.30.318311

Cited by 95 publications

(127 citation statements)

References 68 publications

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“…Overall, the results support observations 21,34 that the role of the SARS-CoV-2 S1/S2 site is to expand viral tropism to lung cells. Cleaved S1/S2 site is crucial for the SARS-CoV-2 to be subsequently cleaved at the S2’ location by TMPRSS2 for immediate plasma membrane entry in respiratory cells.…”

Section: Resultssupporting

confidence: 89%

“…Cleaved S1/S2 site is crucial for the SARS-CoV-2 to be subsequently cleaved at the S2’ location by TMPRSS2 for immediate plasma membrane entry in respiratory cells. Without the S1/S2 pre-cleavage, SARS-CoV-2 would be endocytosed, and due to low cathepsin L expression in respiratory endosomes 10 , coupled with expression of antiviral restriction factors in endosomes 34 , SARS-CoV-2 would not effectively infect via respiratory endosome. If SARS-CoV-2 is infecting TMPRSS2 negative cells, it can utilize endosomal cathepsin L, an ubiquitous protease generally found throughout mammalian cells 35 , to activate the S protein 20 , though at undetermined sites.…”

Section: Resultsmentioning

confidence: 99%

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Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

Tang

Jaimes

Bidon

et al. 2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2’ site is known to activate the S protein for viral entry, which can occur at either the cell plasma membrane or the endosomal membrane. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which expands viral tropism to lung cells. Here, we analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the implications of SARS-CoV-2’s unique insert on its origin. We also utilized viral pseudoparticles to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for plasma membrane entry into Calu-3 cells, a model lung epithelial cell line, but not for endosomal entry Vero E6 cells, a model cell culture line, and that other proteases in addition to furin are responsible for processing SARS-CoV-2 S1/S2.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

Tang

Jaimes

Bidon

et al. 2020

Preprint

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“…In addition, SARS-CoV-2 passaging in Vero cells commonly leads to substitutions or deletions in the S1/S2 cleavage loop (30)(31)(32)(33)(34)(35)(36). In animal models, these viruses exhibit reduced transmission (37) or virulence (38). Mutant viruses bearing substitutions or deletions in the S1/S2 loop are rarely detected in humans (33,37,39) (Suppl.…”

Section: Introductionmentioning

confidence: 99%

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

Laporte

Stevaert

Raeymaekers

et al. 2020

Preprint

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The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33°C instead of 37°C. Even stronger preference for the upper airway temperature of 33°C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37°C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37°C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology.SIGNIFICANCE STATEMENTThe rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33°C) or lower (37°C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.

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“…The inoculum was then removed and cells maintained as described above. 24 h post infection, the culture supernatants were collected and quanti ed by TCID 50 assay on GMK Vero E6 cells as described previously 11 . Serial dilutions of the virus (in serum-free DMEM) were added in 96-well plates and cells were left for 4-5 days before they were xed with 2x crystal violet solution and analysed.…”

Section: Sars-cov-2 Live Virus Infection Studiesmentioning

confidence: 99%

“…the proteases nsp3 and nsp5) proteins 10 . Cellular entry of SARS-CoV-2 requires host proteins expressed on the epithelial cell surface, most essential are Transmembrane Serine Protease 2 (TMPRSS2) and Angiotensin-Converting Enzyme 2 (ACE2) [11][12][13] . TMPRSS2 cleaves and primes the viral Spike (or S) protein; this facilitates fusion of the viral and host membranes [14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

Enzalutamide, a prostate cancer therapeutic, downregulates TMPRSS2 in lung and reduces cellular entry of SARS-CoV-2

Leach

Mohr

Giotis

et al. 2021

Preprint

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The COVID-19 pandemic, caused by the novel human coronavirus SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. To determine therapeutic potential, we assessed uptake of SARS-CoV-2 Spike protein pseudotyped lentivirus and live SARS-CoV-2 into human lung cells and saw a significant reduction in viral entry and infection upon treatment with the antiandrogens enzalutamide and bicalutamide. In support of this new experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

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

Cited by 95 publications

References 68 publications

Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

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

Enzalutamide, a prostate cancer therapeutic, downregulates TMPRSS2 in lung and reduces cellular entry of SARS-CoV-2

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