Role of Q675H Mutation in Improving SARS-CoV-2 Spike Interaction with the Furin Binding Pocket

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants’ evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.

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Phylogenetic analysis and in silico studies link spike Q675H mutation to SARS-CoV-2 adaptive evolution

10.1101/2021.10.27.466055 ◽

2021 ◽

Author(s):

Anna Bertelli ◽

Pasqualina D'Ursi ◽

Giovanni Campisi ◽

Serena Messali ◽

Maria Milanesi ◽

...

Keyword(s):

Viral Entry ◽

In Silico ◽

Cleavage Site ◽

Parallel Evolution ◽

Conformational Space ◽

Furin Cleavage ◽

Fitness Advantage ◽

Furin Cleavage Site ◽

In Silico Studies ◽

Arginine Residues

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region delimiting the conformational space of the highly flexible loop containing the furin cleavage site. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was found to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation is likely to confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we show an ongoing increase in the occurrence of Q675H spike mutation in the most common SARS-CoV-2 variants of concern (VOC). This finding highlights that, VOC are still evolving and start acquiring the Q675H mutation. At the same time, it suggests that our hypothesis of fitness advantage prompted by Q675H could be concrete.

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Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

10.1101/2020.02.19.956581 ◽

2020 ◽

Cited By ~ 91

Author(s):

Alexandra C. Walls ◽

Young-Jun Park ◽

M. Alexandra Tortorici ◽

Abigail Wall ◽

Andrew T. McGuire ◽

...

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Target Cells ◽

Furin Cleavage ◽

Humoral Immune ◽

Binding Domains ◽

Furin Cleavage Site ◽

Recent Emergence ◽

Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

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The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2

Journal of Virology ◽

10.1128/jvi.02422-20 ◽

2021 ◽

Vol 95 (9) ◽

Cited By ~ 4

Author(s):

Helena Winstone ◽

Maria Jose Lista ◽

Alisha C. Reid ◽

Clement Bouton ◽

Suzanne Pickering ◽

...

Keyword(s):

Viral Entry ◽

Cleavage Site ◽

Type I Interferons ◽

Spike Protein ◽

Type I ◽

Furin Cleavage ◽

Independent Manner ◽

Type I Ifns ◽

Furin Cleavage Site ◽

Potent Inhibition

ABSTRACT The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs. IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.

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SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint

10.20944/preprints202110.0080.v2 ◽

2021 ◽

Author(s):

Antonio R. Romeu

Keyword(s):

Middle East ◽

Markov Model ◽

Cleavage Site ◽

Spike Protein ◽

Cleavage Sites ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Arginine Residues ◽

Codon Pair ◽

Shed Light

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

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A 20 Residues Motif Delineates the Furin Cleavage Site and its Physical Properties May Influence Viral Fusion

Biochemistry Insights ◽

10.4137/bci.s2049 ◽

2009 ◽

Vol 2 ◽

pp. BCI.S2049 ◽

Cited By ~ 14

Author(s):

Sun Tian

Keyword(s):

Amino Acids ◽

Physical Properties ◽

Cleavage Site ◽

Binding Pocket ◽

Core Region ◽

Polar Regions ◽

Viral Fusion ◽

Furin Cleavage ◽

The Core ◽

Furin Cleavage Site

Furin is a proprotein convertase that proteolytically cleaves protein precursors to yield functional proteins. Efficient cleavage depends on the presence of a specific sequence motif on the substrate. Currently, the cleavage site motif is described as a four amino acid pattern: R-X-[K/R]-R⇓. However, not all furin cleavage recognition sites can be described by this pattern and not all R-X-[K/R]-R⇓ sites are cleaved by furin. Since many furin substrates are involved in the pathogenesis of viral infection and human diseases, it is important to accurately characterize the furin cleavage site motif. In this study, the furin cleavage site motif was characterized using statistical analysis. The data were interpreted within the 3D crystal structure of the furin catalytic domain. The results indicate that the furin cleavage site motif is comprised of about 20 residues, P14-P6′. Specific physical properties such as volume, charge, and hydrophilicity are required at specific positions. The furin cleavage site motif is divided into two parts: 1) one core region (8 amino acids, positions P6-P2′) packed inside the furin binding pocket; 2) two polar regions (8 amino acids, positions P7–P14; and 4 amino acids, positions P3′-P6′) located outside the furin binding pocket. The physical properties of the core region contribute to the binding strength of the furin substrate, while the polar regions provide a solvent accessible environment and facilitate the accessibility of the core region to the furin binding pocket. This furin cleavage site motif also revealed a dynamic relationship linking the evolution of physical properties in region P1′-P6′ of viral fusion peptides, furin cleavage efficacy, and viral infectivity.

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Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

PLoS Pathogens ◽

10.1371/journal.ppat.1009212 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1009212 ◽

Cited By ~ 1

Author(s):

Tianling Ou ◽

Huihui Mou ◽

Lizhou Zhang ◽

Amrita Ojha ◽

Hyeryun Choe ◽

...

Keyword(s):

Viral Entry ◽

Cathepsin L ◽

Mechanistic Explanation ◽

Furin Cleavage ◽

Culture Studies ◽

S Protein ◽

Furin Cleavage Site ◽

Endosomal Acidification ◽

S Proteins

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

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The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

10.1101/2020.12.19.423592 ◽

2020 ◽

Author(s):

Helena Winstone ◽

Maria Jose Lista ◽

Alisha Reid ◽

Suzanne Pickering ◽

Katie J Doores ◽

...

Keyword(s):

Viral Entry ◽

Cleavage Site ◽

Type I Interferons ◽

Type I ◽

Furin Cleavage ◽

Independent Manner ◽

Type I Ifns ◽

Furin Cleavage Site ◽

Late Endosomes ◽

Potent Inhibition

ABSTRACTThe cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein (S). The presence of a polybasic cleavage site in SARS-CoV-2 S at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the S precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons, and more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2 and the degree of this restriction is governed by route of viral entry. Removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH- and cathepsin-dependent in late endosomes where, like SARS-CoV-1 S, it is more sensitive to IFITM2 restriction. Furthermore, we find that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 S may reduce viral replication through the activity of type I IFNs.IMPORTANCEThe furin cleavage site in the S protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2, and that the sensitivity is exacerbated by deletion of the furin cleavage site which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus may represent a potential therapeutic target for COVID-19 treatment development.

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Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

10.1101/2020.07.22.216150 ◽

2020 ◽

Cited By ~ 4

Author(s):

Tianling Ou ◽

Huihui Mou ◽

Lizhou Zhang ◽

Amrita Ojha ◽

Hyeryun Choe ◽

...

Keyword(s):

Cell Culture ◽

Clinical Trials ◽

Viral Infection ◽

Viral Entry ◽

Cleavage Site ◽

Cathepsin L ◽

Furin Cleavage ◽

S Protein ◽

Furin Cleavage Site

AbstractHydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.Author SummaryThe novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.

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Trypsin Enhances SARS-CoV-2 Infection by Facilitating Viral Entry

10.21203/rs.3.rs-919057/v1 ◽

2021 ◽

Author(s):

Yeeun Kim ◽

Guehwan Jang ◽

Duri Lee ◽

Nara Kim ◽

Jeong Won Seon ◽

...

Keyword(s):

Viral Entry ◽

Cultured Cells ◽

Tissue Injury ◽

Furin Cleavage ◽

S Protein ◽

Endosomal Membrane ◽

Furin Cleavage Site ◽

Target Organs ◽

Syncytia Formation

Abstract Coronavirus infects the cell by cytoplasmic or endosomal membrane fusion driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and S2′ site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we showed that trypsin mediated the enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 entered the cells via either a non-endosomal or an endosomal fusion pathway depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection efficiency than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger high replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytia formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin for the cell-adapted SARS-CoV-2 strains. During 50 serial passages without trypsin addition, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but with several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that S1/S2 FCS is essential for S protein proteolytic priming and fusion activity for SARS-CoV-2 entry but not for SARS-CoV-2 replication. Our data can contribute to the improvement of SARS-CoV-2 production to develop vaccines or antivirals and motivate further investigations into the explicit functions of cell adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

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SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint

10.20944/preprints202110.0080.v1 ◽

2021 ◽

Author(s):

Antonio R. Romeu

Keyword(s):

Markov Chain ◽

Middle East ◽

Cleavage Site ◽

Spike Protein ◽

Furin Cleavage ◽

First Order ◽

Furin Cleavage Site ◽

Order Markov Chain ◽

Arginine Residues ◽

Shed Light

At present, the polybasic furin cleavage site on the spike glycoprotein of the SARS-CoV-2 is still a missing link. Remarkably, the two arginine residues this of site are encoded by the CGG arginine codon, which is rare in Betacoronavirus proteins. Arginine dimers are common at viral furin sites, but are not CGG-CGG encoded. The question is: Is that genetic footprint, encoding arginine pairs, unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers that exist in Betacoronavirus proteins. As main result, a set of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) have the CGG-CGG encoded arginine pair in the spike protein polybasic furin cleavage site. In addition, CGG-CGG encoded arginine pairs were also found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the presence probability of CGG-CGG arginine-arginine in Betacoronavirus, a First-Order Markov Chain was defined. It is highly unlikely to find it in betacoronaviruses wildlife, but it is there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine dimer in the S1/S2 cleavage site.

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