key: cord-0735137-l3wt7lqo
authors: Stevaert, Annelies; Van Berwaer, Ria; Raeymaekers, Valerie; Laporte, Manon; Naesens, Lieve
title: Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13
date: 2022-01-27
journal: bioRxiv
DOI: 10.1101/2022.01.26.477969
sha: 29cfacab6b53949258bc169c8962909997b1a619
doc_id: 735137
cord_uid: l3wt7lqo

The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2’ motif which, during virus entry, requires cleavage by a cell surface protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2’ motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the S2’ motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689) and also the reverse effect was seen. This swap had no impact on TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by TMPRSS2. Our findings are the first to demonstrate which S2’ residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2’ motif changes as they may appear in new SARS-CoV-2 variants. IMPORTANCE Since the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2’ cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2’ site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2’ site variations as soon as they are detected during variant surveillance.

Since the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning in a direct manner. Remarkably, though located in a strong immunogenic region, the S2´ cleavage motif has, thus far, remained highly conserved. This suggests that its amino acid sequence is critical for spike activation by airway proteases. To investigate this, we assessed which S2´ site mutations affect processing by TMPRSS2 and TMPRSS13, two main activators of the SARS-CoV-2 spike. Being the first in its kind, our study will help to assess the biological impact of S2´ site variations as soon as they are detected during variant surveillance.

SARS-CoV-2, human coronavirus 229E, spike protein, mutation, protease, cleavage,

Since the start of the COVID-19 pandemic, SARS-CoV-2 is undergoing worldwide selection with frequent appearance of new variants. The variability in the viral spike (S) antigen is linked to immune evasion but also affects the functioning of S in virus replication and transmission. For instance, substitution D614G, which arose in March 2020 to soon become dominant, increases S protein stability (1, 2) and virus transmission (3, 4) . Mutation P681R, present in the delta and kappa variants and located adjacent to the S1/S2 furin recognition motif (RRAR), enhances spike fusogenicity and virus pathogenicity in hamsters (5) . The reverse is seen for the omicron variant (6, 7) . Knowing which residues in the functional spike motifs are essential or not, can help to assess the impact of new variations as they emerge. In this report, we focus on the S2´ motif.

Cleavage of the SARS-CoV-2 S1/S2 site facilitates cleavage at the S2´ site, a process essential to release the fusion peptide. Two efficient S2´ activators are TMPRSS2 (8) and TMPRSS13 (2, 9, 10) which are both expressed in respiratory tissue (11) . In a SARS-CoV-2 mouse model, TMPRSS2 knockout resulted in less lung pathology and lower virus titers, although the virus was still able to replicate in the lungs (12) . In human airway-derived Calu-3 cells, knockdown of TMPRSS2 reduced virus replication dramatically (2, 13) , yet also TMPRSS13 knockdown had significant effect (2) . The activating role of TMPRSS2, but not that of TMPRSS13, was confirmed in human airway organoids (14) . Hence, the relative contribution of these two proteases in SARS-CoV-2 infection remains unclear.

Low variability and K/R abundance in the S2´ site. Within the SARS-CoV-2 S2´ motif (811-KPSKR-815), the scissile R 815 residue is flanked by a second basic (K 814 ) residue. The motif lies in a strong epitope (15, 16) for which the antibody titers seem correlated with COVID-19 disease severity (17) . Despite this high immunogenicity, the S2´ motif exhibits strikingly low variability. When we analyzed the ~6.7 million spike sequences in the GISAID database (Fig. 1A) , all three basic residues in the S2´ motif (= K 811 , K 814 and R 815 ) proved highly dominant, being present in >99.99% of the sequences. This is less surprising for R 815 (in only a few cases substituted by K), the presumed scissile residue (18) . P 812 and S 813 are somewhat more tolerant towards variation, consistent with the presence of T 813 in the SARS-CoV spike (19) .

Mutations at the S2´ motif do not affect spike expression or S1/S2 cleavage.

The high conservation of the SARS-CoV-2 KxxKR motif suggests that its sequence is essential for spike activation. Strikingly, a dibasic scissile motif is missing in all five endemic less pathogenic human coronaviruses (HCoVs) including HCoV-229E (19) , while MERS-CoV bears an xRxxR motif (Fig. 1B) . Hence, we designed a series of S2´-mutated S proteins (abbreviated SARS-2-S, MERS-S and 229E-S; Fig. 1B ) in which we swapped the motifs from SARS-2-S and 229E-S or introduced or removed a Lys (K) at P2 or an Arg (R) at P4 (since MERS-S contains a basic residue at P4 but not P5). S-bearing MLV-pseudoparticles were produced in HEK293T cells, pelleted down and analyzed for spike levels and cleavage by western blot (Fig. 2A) . All S2´mutant pseudovirions contained similar S protein levels as the respective WT (Fig.   2B ). For the SARS-2-S-and MERS-S-pseudoparticles, efficient spike cleavage was seen for all S2´mutants. Even a change of 4 out of 5 residues (= SARS-2-S-Mut3) had no effect on spike expression or S1/S2 cleavage. For 229E-S, the WT and mutants contained similar levels of several cleavage products.

SARS-2-S containing the S2´ motif of 229E-S loses TMPRSS13 cleavability. We next conducted an entry assay to measure S2´ activation by TMPRSS2 and TMPRSS13. The S-pseudoviruses were transduced into HEK293T cells which were transfected one day earlier with TMPRSS2-, TMPRSS13-or empty plasmid, plus the cognate virus receptor ( Fig. 2A) . Transduction was performed in the presence of E64d to shut off the endosomal route in which S is activated by cathepsin B/L instead of cell surface proteases (20) . For MERS-S, activation by TMPRSS2 was significantly yet only 3.4-fold reduced when the motif contained a third basic residue (Mut1). Substituting the R at P4 (Mut2) had no (TMPRSS2) or only minor (TMPRSS13) influence.

During the above experiments, equal amounts of TMPRSS2-and TMPRSS13plasmid DNA were applied for HEK293T cell transfection. Dot blot analysis demonstrated that this generated comparable protein levels for both proteases (Fig.   2D ). This excluded the possibility that low expression of TMPRSS13 might be the reason for poor activation of some pseudoviruses.

Discussion. This study anticipates on the relevance of S2´ site changes as they may appear in new SARS-CoV-2 variants. Since the S2´ motif lies in a strong epitope (15, 17) , the virus might acquire mutations in this region to evade immunity. The fact that such variations are, thus far, rarely detected could mean that the sequence of this K/R rich motif is crucial for SARS-2-S activation by cell surface proteases. Our study is the first to address this topic. Clearly, residue K 814 is important for TMPRSS13 but not TMPRSS2. This basic P2 residue differentiates SARS-2-S from 229E-S (and the spikes of all other low pathogenic HCoVs), which otherwise share a basic P5 residue.

Making the motif even more basic by addition of an R at P4, causes a more dramatic reduction for TMPRSS2 than TMPRSS13. This aligns with data that basic residues at P2-P3-P4 are not favored by TMPRSS2 (22) . Reciprocally, the preference of TMPRSS13 for a basic motif concurs with its activity on multibasic hemagglutinins of highly pathogenic avian influenza viruses (23) . Combined with the presence of TMPRSS13 in human lung tissue (24) , this raises the hypothesis that loss of TMPRSS13 cleavability, e.g. by mutation of K 814 , might reduce replication of SARS-CoV-2 in the lungs. Though limited to a few mutations, our findings will help to estimate the impact of S2´ site changes observed during variant surveillance. 

The authors thank S. Pöhlmann and M. Hoffmann for the kind gift of plasmid materials.

There are no relevant financial or non-financial competing interests to report.

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

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

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

SARS-CoV-2 spike D614G change enhances replication and transmission

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

TMPRSS11D and TMPRSS13 activate the SARS-CoV-2 spike protein

Hemagglutinin cleavability, acid stability, and temperature dependence optimize influenza B virus for replication in human airways

Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide

Böttcher-Friebertshäuser E. 2020. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients

Functional analysis of human and feline coronavirus crossreactive antibodies directed against the SARS-CoV-2 fusion peptide

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern

Binding of SARS-CoV-2 fusion peptide to host endosome and plasma membrane

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

Coronavirus membrane fusion mechanism offers a potential target for antiviral development

TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium

The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis

Novel type II transmembrane serine proteases, MSPL and TMPRSS13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication

Cloning and expression of novel mosaic serine proteases with and without a transmembrane domain from human lung

Data, disease and diplomacy: GISAID's innovative contribution to global health