scholarly journals The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells

2020 ◽  
Author(s):  
Anna Z. Mykytyn ◽  
Tim I. Breugem ◽  
Samra Riesebosch ◽  
Debby Schipper ◽  
Petra B. van den Doel ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Highly Pathogenic ◽  
Remarkable Feature ◽  
Health Crisis ◽  
Human Airway ◽  
Cell Culture Systems ◽  
Serine Protease Activity ◽  
Airway Cells

AbstractAfter the SARS-CoV outbreak in 2003, a second zoonotic coronavirus named SARS-CoV-2, emerged late 2019 in China and rapidly caused the COVID-19 pandemic leading to a public health crisis of an unprecedented scale. Despite the fact that SARS-CoV-2 uses the same receptor as SARS-CoV, transmission and pathogenesis of both viruses seem to be quite distinct. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site, which is absent in the SARS-CoV spike. The viral spike protein not only attaches to the entry receptor, but also mediates fusion after cleavage by host proteases. Here, we report that the SARS-CoV-2 spike multibasic cleavage site increases infectivity on differentiated organoid-derived human airway cells. Compared with SARS-CoV, SARS-CoV-2 entered faster into the lung cell line Calu-3, and more frequently formed syncytial cells in differentiated organoid-derived human airway cells. Moreover, the multibasic cleavage site increased entry speed and plasma membrane serine protease usage relative to endosomal entry using cathepsins. Blocking serine protease activity using the clinically approved drug camostat mesylate effectively inhibited SARS-CoV-2 entry and replication in differentiated organoid-derived human airway cells. Our findings provide novel information on how SARS-CoV-2 enters relevant airway cells and highlight serine proteases as an attractive antiviral target.Significance StatementHighly pathogenic coronaviruses have spilled from animals to humans three times in the past two decades. Late 2019, SARS-CoV-2 emerged in China and was declared a pandemic by March 2020. The other two highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, emerged in 2002 and 2012, respectively, but did not attain sustained human-to-human transmission. Given the high diversity of coronaviruses in animals, urbanization and increased air travel, future coronavirus pandemics are likely to occur intermittently. Identifying which factors determine pandemic potential and pathogenicity are therefore of key importance to global health. Additionally, there is an urgent need to rapidly translate fundamental knowledge to the clinic, a process that is expedited through the use of relevant cell culture systems.

scholarly journals SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Anna Z Mykytyn ◽  
Tim I Breugem ◽  
Samra Riesebosch ◽  
Debby Schipper ◽  
Petra B van den Doel ◽  
...  
Keyword(s):  
Serine Protease ◽  
Viral Entry ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Spike Protein ◽  
Remarkable Feature ◽  
Human Airway ◽  
Entry Strategy ◽  
Entry Speed

Coronavirus entry is mediated by the spike protein which binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells, and more frequently formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.

scholarly journals Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

2021 ◽  
Author(s):  
Mart M. Lamers ◽  
Anna Z. Mykytyn ◽  
Tim I. Breugem ◽  
Yiquan Wang ◽  
Douglas C. Wu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Culture Conditions ◽  
Virus Propagation ◽  
Human Airway ◽  
Culture Adaptation ◽  
Propagation Methods ◽  
Transformed Cell Lines ◽  
Airway Cells

AbstractVirus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

scholarly journals Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mart M Lamers ◽  
Anna Z Mykytyn ◽  
Tim I Breugem ◽  
Yiquan Wang ◽  
Douglas C Wu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Culture Conditions ◽  
Virus Propagation ◽  
Human Airway ◽  
Culture Adaptation ◽  
Propagation Methods ◽  
Transformed Cell Lines ◽  
Airway Cells

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

scholarly journals Multifaceted Analyses of Epidermal Serine Protease Activity in Patients with Atopic Dermatitis

2020 ◽  
Vol 21 (3) ◽  
pp. 913 ◽  
Author(s):  
Hayato Nomura ◽  
Mutsumi Suganuma ◽  
Takuya Takeichi ◽  
Michihiro Kono ◽  
Yuki Isokane ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Serine Protease ◽  
Protease Activity ◽  
Serine Proteases ◽  
Immunoglobulin E ◽  
Total Serum ◽  
Tape Stripping ◽  
Blood Eosinophil ◽  
Peripheral Blood Eosinophil ◽  
Serine Protease Activity

The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals (n = 46) and AD patients (n = 63) by tape stripping and then measuring the trypsin- and chymotrypsin-like serine protease activity. We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC’s serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.

scholarly journals Therapeutic targeting of coronavirus spike glycoprotein priming

2020 ◽  
Author(s):  
Maurizio Pellecchia ◽  
Elisa Barile ◽  
Carlo Baggio ◽  
Luca Gambini ◽  
Sergey A. Shiryaev ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Inhibitors ◽  
Serine Proteases ◽  
Viral Proteins ◽  
Exact Nature ◽  
Cleavage Sites ◽  
Serine Protease Inhibitors ◽  
Spike Glycoprotein ◽  
Highly Pathogenic

Abstract Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2’) cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species, and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2’ cleavage sites of their spike glycoprotein. However there remains some confusion on the relative roles of the possible serine-proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing virulence. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of the additional mutations in the S-protein priming sequence in coronaviruses.

scholarly journals 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

2021 ◽  
Author(s):  
Jonathan C. Brown ◽  
Daniel H. Goldhill ◽  
Jie Zhou ◽  
Thomas P. Peacock ◽  
Rebecca Frise ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cleavage Site ◽  
Vero Cells ◽  
Spike Protein ◽  
Airway Epithelial ◽  
Post Vaccination ◽  
Human Airway ◽  
Rapid Spread ◽  
The Uk ◽  
Airway Cells

AbstractLineage 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.

scholarly journals TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

2020 ◽  
Vol 3 (9) ◽  
pp. e202000786 ◽  
Author(s):  
Dorothea Bestle ◽  
Miriam Ruth Heindl ◽  
Hannah Limburg ◽  
Thuy Van Lam van ◽  
Oliver Pilgram ◽  
...  
Keyword(s):  
Serine Protease ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Severe Disease ◽  
Acute Infection ◽  
Airway Epithelial Cells ◽  
Surface Glycoprotein ◽  
Proteolytic Activation ◽  
Human Airway ◽  
Airway Cells

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

scholarly journals Protean proteases: at the cutting edge of lung diseases

2017 ◽  
Vol 49 (2) ◽  
pp. 1501200 ◽  
Author(s):  
Clifford Taggart ◽  
Marcus A. Mall ◽  
Gilles Lalmanach ◽  
Didier Cataldo ◽  
Andreas Ludwig ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Protease Activity ◽  
Serine Proteases ◽  
Lung Diseases ◽  
Serine Protease Inhibitor ◽  
Matrix Metalloproteases ◽  
Integrative Biology ◽  
Therapeutic Tool ◽  
Serine Protease Activity

Proteases were traditionally viewed as mere protein-degrading enzymes with a very restricted spectrum of substrates. A major expansion in protease research has uncovered a variety of novel substrates, and it is now evident that proteases are critical pleiotropic actors orchestrating pathophysiological processes. Recent findings evidenced that the net proteolytic activity also relies upon interconnections between different protease and protease inhibitor families in the protease web.In this review, we provide an overview of these novel concepts with a particular focus on pulmonary pathophysiology. We describe the emerging roles of several protease families including cysteine and serine proteases.The complexity of the protease web is exemplified in the light of multidimensional regulation of serine protease activity by matrix metalloproteases through cognate serine protease inhibitor processing. Finally, we will highlight how deregulated protease activity during pulmonary pathogenesis may be exploited for diagnosis/prognosis purposes, and utilised as a therapeutic tool using nanotechnologies.Considering proteases as part of an integrative biology perspective may pave the way for the development of new therapeutic targets to treat pulmonary diseases related to intrinsic protease deregulation.

scholarly journals Serine proteases in the extracellular preparations of Phytophthora infestans: does their presence relate to the aggressiveness of the pathogen?

2002 ◽  
Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽  
pp. S102-S103
Author(s):  
J. Hamill ◽  
C. Selby ◽  
L.R. Cooke
Keyword(s):  
Phytophthora Infestans ◽  
Serine Protease ◽  
Ethyl Ester ◽  
Proteolytic Activity ◽  
Protease Activity ◽  
Serine Proteases ◽  
Daily Basis ◽  
Serine Protease Activity ◽  
Zoospore Suspension

In this study the aggressiveness of nine isolates of P. infestans was determined using detached leaflets from cultivars Bintje and Stirling. The growth of the isolates on the leaflets was recorded on a daily basis, for seven days, and an assessment of their aggressiveness could then be made. Extracellular preparations (ECPs) from the zoospore suspension of each isolate were used as a source of proteolytic activity. The ECPs were found to contain a level of serine protease activity using BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester) as a substrate and recording the absorbance at 256 nm. The possible relationship between the serine protease activity and the aggressiveness of the isolate is discussed.

scholarly journals Overhydration Measured by Bioimpedance Spectroscopy and Urinary Serine Protease Activity Are Risk Factors for Progression of Chronic Kidney Disease

2020 ◽  
Vol 45 (6) ◽  
pp. 955-968
Author(s):  
Anja Schork ◽  
Bernhard N. Bohnert ◽  
Nils Heyne ◽  
Andreas L. Birkenfeld ◽  
Ferruh Artunc
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Serine Protease ◽  
Protease Activity ◽  
Serine Proteases ◽  
Cox Regression ◽  
Bioimpedance Spectroscopy ◽  
Operating Characteristics ◽  
Cutoff Values ◽  
Serine Protease Activity

<b><i>Background:</i></b> Overhydration (OH) is common in chronic kidney disease (CKD) and might be related to the excretion of urinary serine proteases. Progression of CKD is associated with proteinuria; however, the interrelations of urinary serine proteases, OH, and progression of CKD remain unclear. <b><i>Methods:</i></b> In <i>n</i> = 179 patients with stable nondialysis-dependent CKD of all stages, OH was measured using bioimpedance spectroscopy (Body Composition Monitor; Fresenius), and urinary serine protease activity was determined using the peptide substrate S-2302. After a median follow-up of 5.9 (IQR: 3.9–6.5) years, progression to end-stage renal disease (ESRD) was analyzed retrospectively. <b><i>Results:</i></b> OH correlated with baseline MDRD-eGFR, urinary albumin creatinine ratio (ACR), and urinary aprotinin-sensitive serine protease activity. Progression to ESRD occurred in <i>n</i> = 33 patients (19%) and correlated with OH and urinary serine protease activity as well as MDRD-eGFR and ACR. Patients were divided into 2 groups determined by cutoff values from receiver operating characteristics for MDRD-eGFR (32 mL/min/1.73 m<sup>2</sup>), ACR (43 mg/g creatinine), urinary serine protease activity (0.9 RU/g creatinine), and OH (1 L/1.73 m<sup>2</sup>). Across these cutoff values, Kaplan-Meier curves for renal survival showed significant separations of the groups. In Cox regression adjusted for MDRD-eGFR, ACR, P-NT-pro-BNP, systolic blood pressure, and diabetes mellitus, patients with OH &#x3e;1 L/1.73 m<sup>2</sup> had a 3.32 (95% CI: 1.26–8.76)-fold higher risk for progression to ESRD. <b><i>Conclusions:</i></b> Our results corroborate that OH detected by bioimpedance spectroscopy in CKD patients is an independent risk factor for progression to ESRD in addition to GFR and albuminuria. Urinary serine protease activity is associated with OH and progression of CKD and provides a possible underlying mechanism.

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