key: cord-291991-on70zzn0
authors: Jaimes, Javier A.; Millet, Jean K.; Whittaker, Gary R.
title: Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 site
date: 2020-05-28
journal: iScience
DOI: 10.1016/j.isci.2020.101212
sha: 
doc_id: 291991
cord_uid: on70zzn0

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread to the entire world within a few months. The origin of SARS-CoV-2 has been related to the lineage B Betacoronavirus SARS-CoV and SARS-related coronaviruses found in bats. Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct 4 amino acid insert within the spike (S) protein (underlined, SPRRAR↓S), at the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this insert appears to be a distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases. We discuss these findings in the context of the origin of SARS-CoV-2, viral stability and transmission.

Since December 2019, human infections by a novel coronavirus (CoV) named severe acute respiratory 29 syndrome coronavirus 2 (SARS-CoV-2) have rapidly spread globally from an initial outbreak of severe 30 pneumonia centered on Wuhan, Hubei Province, China. The virus is the etiological agent of an infectious 31 respiratory disease termed coronavirus disease 19 (COVID-19). On March 11 th 2020, due to its alarming 32 spread and severity across most countries globally the WHO characterized COVID-19 as a pandemic. 2009), found in the S2 fusion domain immediately upstream of the fusion peptide-whose functional 57 role is more directly comparable to that of influenza virus HA cleavage site (Steinhauer, 1999) . 

Here we provide context and clarify the role of the novel SARS-CoV-2 S1/S2 cleavage site in virus 90 emergence and infection, and perform a direct assessment of the proteases cleaving this site by use of 91 biochemical assays.

To directly address the proteases cleaving the SARS-CoV-2 S1/S2 site, we used a biochemical peptide 95 cleavage assay (Jaimes et al., 2019), which was previously used to screen emerging influenza viruses 96 (Straus and Whittaker, 2017) . This assay was also successfully used to study the functional impact of 98 Millet et al., 2016) . The peptide sequences used here were HTVSLLRSTSQ (SARS-CoV S1/S2) and 99 TNSPRRARSVA (SARS-CoV-2 S1/S2). We tested a range of proteases likely to be involved in spike protein 100 processing-the proprotein convertases furin and PC1, trypsin and the type II transmembrane serine 101 protease (TTSP) matriptase, as well as cathepsins B and L. As predicted, furin cleaves SARS-CoV-2, but 102 not SARS-CoV ( fig. 1) . However, in addition to furin, other proteases also cleaved SARS-CoV-2 much 103 more readily than SARS-CoV. PC1 showed a similar cleavage pattern but with lower efficiency than for 104 furin. Trypsin cleaved both peptides, but was over 4-fold more efficient for SARS-CoV-2. Both the TTSP 105 matriptase and cathepsin B did not cleave SARS-CoV at all, but were highly active on SARS-CoV-2. The 106 only protease that showed more cleavage on SARS-CoV compared to SARS-CoV-2 was cathepsin L. Our 107 data demonstrate that the S1/S2 site of SARS-CoV-2 S is efficiently cleaved by a wide range of proteases, 108 not only furin. The comparative data with SARS-CoV S1/S2 site reveals that the acquisition of the 4 109 amino acid insert distinctively broadens the activating protease repertoire of the SARS-CoV-2 S1/S2 110 cleavage site to all major classes of proteolytic enzymes known to potentially activate coronavirus S 111 proteins.

The COVID-19 pandemic represents a global public health emergency. The origin of the causative agent 114 of the disease, SARS-CoV-2 remains a mystery. Previous analyses have revealed distinctive features 115 within the genome of SARS-CoV-2. In particular, a 4 amino acid insert was found within the S1/S2 site of 116 the S glycoprotein. This finding has garnered interest because of the possible introduction of a furin 117 recognition motif at the S1/S2 site. Our study aimed at clarifying the functional role of the S1/S2 118 cleavage site for SARS-CoV-2. We provide direct biochemical evidence that the site S1/S2 is recognized 119 and cleaved by furin. However, other proteases such as PC1 another member of the PC family of 120 proteases, trypsin-like proteases, and cathepsins can all efficiently recognize and cleave SARS-CoV-2 121 S1/S2 cleavage site. These data confirm earlier findings showing the SARS-CoV-2 entry was dependent, 122 at least in part, on lysosomal cathepsins and the cell surface-expressed TTSPs such as TMPRSS2 7 (Hoffmann et al., 2020b) . Interestingly, the comparative assessment we present with SARS-CoV shows 124 that the S1/S2 insertion SARS-CoV-2 has acquired substantially expands its proteolytic activation profile, 125 with potential activation from a wide variety of proteases. Further experiments will include the 126 evaluation of the protease cleavage of the complete trimeric form of the SARS-CoV-2 S protein.

Another feature of the S1/S2 junction that has been noted for SARS-CoV-2 is the presence of a leading 

We favor a model whereby the novel S1/S2 insert allows furin cleavage but also reflects a more 141 enhanced exposure of a critical cleavage site to TTSPs such as TMPRSS2 and matriptase, as well as 142 cathepsin B, an activation mechanism that is not necessarily through the action of furin itself. Overall, 143 the novel S1/S2 insert is likely to enhance spike protein cleavage by multiple proteases beyond that for 144 other SARS-like viruses (e.g. containing the sequence SR↓S). The role of the fusion peptide-proximal S2' 145 site remains to be evaluated, but its sequence (PDPSKPSKR↓SFIEDLLF) is not distinctly different from 146 other SARS-like viruses (Jaimes et al., 2020) . We also propose that the SARS-CoV-2 S1/S2 cleavage site 8 likely arose by mutation/recombination as for influenza H9 viruses, rather than by polymerase slippage 148 as for the more widely appreciated polybasic sites found in H7 and H5 HPAI, and that the term 149 "polybasic site" is a misnomer for SARS-CoV-2. This concept has recently been reinforced by the finding 150 of a bat coronavirus (BatCoV-RmYN02) closely related to SARS-CoV-2 that has an extended S1/S2 

Fluorogenic peptides mimicking the SARS-CoV and SARS-CoV-2 S1/S2 regions were used to evaluate 163 cleavability by recombinant cellular proteases. Each peptide is 11 amino acids long and harbors the 164 specific S1/S2 sequence for each virus, and cleavage of the peptide by a protease is driven by both the 

The proximal 193 origin of SARS-CoV-2

Activation of the SARS coronavirus spike 195 protein via sequential proteolytic cleavage at two distinct sites

The 198 spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent 199 in CoV of the same clade

A Multibasic Cleavage Site in the 201

Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

SARS-CoV-2 Cell Entry Depends on ACE2 205 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Coronavirus Spike Protein and Tropism 207 Changes

Phylogenetic 209 Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct 210 and Proteolytically Sensitive Activation Loop

A Fluorogenic 213

Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike 214 protein activation

Sequence requirements for cleavage activation of 216 influenza virus hemagglutinin expressed in mammalian cells

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by 220

Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous 221 System

Use of AAScatterPlot tool for monitoring the evolution of 223 the hemagglutinin cleavage site in H9 avian influenza viruses

Mutation in spike protein cleavage site and pathogenesis of feline coronavirus

Host cell proteases: Critical determinants of coronavirus 228 tropism and pathogenesis

A 230 camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion 231 activation properties

Host cell entry of Middle East respiratory syndrome 233 coronavirus after two-step, furin-mediated activation of the spike protein

Genetic Predisposition To Acquire a 237

Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-240 reactivity with SARS-CoV

Localization of Endogenous Furin in Cultured Cell Lines

Role of hemagglutinin cleavage for the pathogenicity of influenza virus

A peptide-based approach to evaluate the adaptability 247 of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

A Novel Activation Mechanism 250 of Avian Influenza Virus H9N2 by Furin

Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

A novel bat coronavirus closely related to SARS-CoV-2 contains natural insertions at 258 the S1/S2 cleavage site of the spike protein

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Fluorogenic peptides derived from SARS-CoV and SARS-CoV-2 spike (S) S1/S2 sites composed of the sequences HTVSLLRSTSQ and TNSPRRARSVA sequences, respectively, and harboring the (7-methoxycoumarin-4-yl)acetyl/2,4-dinitrophenyl (MCA/DNP) FRET pair were synthesized by Biomatik

Recombinant PC1, matriptase, cathepsin B , and cathepsin L were purchased from R&D Systems

U/mL); 25 mM MES, 5 mM CaCl2, 1% (w/v) Brij-35, pH 6.0 for PC1 (diluted to 2.2 ng/μL); PBS for trypsin (diluted to 8 nM); 50 mM Tris, 50 mM NaCl, 0.01% (v/v) Tween® 20, pH 9.0 for matriptase (diluted to 2.2 ng/μL); 25 mM MES, pH 5.0 for cathepsin B (diluted to 2.2 ng/μL); 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0 for cathepsin L (diluted to 2.2 ng/μL) and with the peptide diluted to 50 μM. Reactions were performed at 30 °C in triplicates, and fluorescence emission was measured every minute for 45 min using a SpectraMax fluorometer (Molecular Devices