Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

2021 ◽  
Author(s):  
Mizuki Yamamoto ◽  
Jin Gohda ◽  
Ayako Kobayashi ◽  
Keiko Tomita ◽  
Youko Hirayama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Spike Protein ◽  
Dependent Manner ◽  
Furin Cleavage ◽  
Entry Pathway ◽  
Furin Cleavage Site ◽  
Metalloproteinase Inhibitors ◽  
A Cell ◽  
A Disintegrin And Metalloproteinase ◽  
Human Coronaviruses

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

scholarly journals QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

2021 ◽  
Author(s):  
Michelle N Vu ◽  
Kumari Lokugamage ◽  
Jessica A Plante ◽  
Dionna Scharton ◽  
Bryan A Johnson ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Unusual Feature ◽  
Spike Protein ◽  
Loop Length ◽  
Dependent Manner ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Respiratory Cells

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site (the FCS, loop length, and glycosylation) are required for efficient SARS-CoV-2 replication and pathogenesis. 

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

2021 ◽  
Author(s):  
Alison E Stout ◽  
Qinghua Guo ◽  
Jean K Millet ◽  
Gary R Whittaker
Keyword(s):  
Viral Pathogenesis ◽  
Spike Protein ◽  
Future Research ◽  
Receptor Binding Domain ◽  
Viral Reservoirs ◽  
Furin Cleavage ◽  
Proteolytic Activation ◽  
Furin Cleavage Site ◽  
Unique Aspect ◽  
Genome Level

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

scholarly journals A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2) is the long-sought-after cell-surface hyaluronidase

2017 ◽  
Vol 292 (18) ◽  
pp. 7304-7313 ◽  
Author(s):  
Hayato Yamamoto ◽  
Yuki Tobisawa ◽  
Toshihiro Inubushi ◽  
Fumitoshi Irie ◽  
Chikara Ohyama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dermatan Sulfate ◽  
Transmembrane Protein ◽  
Live Cells ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Ph Optimum ◽  
Hyaluronidase Activity ◽  
A Cell ◽  
Intermediate Size

Hyaluronan (HA) is an extremely large polysaccharide (glycosaminoglycan) involved in many cellular functions. HA catabolism is thought to involve the initial cleavage of extracellular high-molecular-weight (HMW) HA into intermediate-size HA by an extracellular or cell-surface hyaluronidase, internalization of intermediate-size HA, and complete degradation into monosaccharides in lysosomes. Despite considerable research, the identity of the hyaluronidase responsible for the initial HA cleavage in the extracellular space remains elusive. HYAL1 and HYAL2 have properties more consistent with lysosomal hyaluronidases, whereas CEMIP/KIAA1199, a recently identified HA-binding molecule that has HA-degrading activity, requires the participation of the clathrin-coated pit pathway of live cells for HA degradation. Here we show that transmembrane protein 2 (TMEM2), a mammalian homolog of a protein playing a role in zebrafish endocardial cushion development, is a cell-surface hyaluronidase. Live immunostaining and surface biotinylation assays confirmed that mouse TMEM2 is expressed on the cell surface in a type II transmembrane topology. TMEM2 degraded HMW-HA into ∼5-kDa fragments but did not cleave chondroitin sulfate or dermatan sulfate, indicating its specificity to HA. The hyaluronidase activity of TMEM2 was Ca2+-dependent; the enzyme's pH optimum is around 6–7, and unlike CEMIP/KIAA1199, TMEM2 does not require the participation of live cells for its hyaluronidase activity. Moreover, TMEM2-expressing cells could eliminate HA immobilized on a glass surface in a contact-dependent manner. Together, these data suggest that TMEM2 is the long-sought-after hyaluronidase that cleaves extracellular HMW-HA into intermediate-size fragments before internalization and degradation in the lysosome.

scholarly journals Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

2020 ◽  
Author(s):  
Z. Galen WO
Keyword(s):  
Amino Acid ◽  
Cleavage Site ◽  
Spike Protein ◽  
Sequence Motif ◽  
Furin Cleavage ◽  
Host Cell Membrane ◽  
Virus Family ◽  
Prediction Program ◽  
Furin Cleavage Site ◽  
Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

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

2020 ◽  
Author(s):  
Thomas P. Peacock ◽  
Daniel H. Goldhill ◽  
Jie Zhou ◽  
Laury Baillon ◽  
Rebecca Frise ◽  
...  
Keyword(s):  
Virus Entry ◽  
Selective Advantage ◽  
Lung Cells ◽  
Cleavage Sites ◽  
Airway Epithelial ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Epithelial Cultures ◽  
A Cell ◽  
Cell Surface Protease

SummarySARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

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

2021 ◽  
Vol 95 (9) ◽  
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.

scholarly journals Gene of the month: the 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein

2020 ◽  
Vol 73 (7) ◽  
pp. 366-369 ◽  
Author(s):  
Tahir S Pillay
Keyword(s):  
Receptor Binding ◽  
Transmembrane Domain ◽  
Vaccine Development ◽  
Protein S ◽  
Respiratory Illness ◽  
Spike Protein ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Cellular Entry ◽  
Novel Coronavirus

The year 2020 has seen a major and sustained outbreak of a novel betacoronavirus (severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2) infection that causes fever, severe respiratory illness and pneumonia, a disease called COVID-19. At the time of writing, the death toll was greater than 120 000 worldwide with more than 2 million documented infections. The genome of the CoV encodes a number of structural proteins that facilitate cellular entry and assembly of virions, of which the spike protein S appears to be critical for cellular entry. The spike protein guides the virus to attach to the host cell. The spike protein contains a receptor-binding domain (RBD), a fusion domain and a transmembrane domain. The RBD of spike protein S binds to Angiotensin Converting Enzyme 2 (ACE2) to initiate cellular entry. The spike protein of SARS-CoV-2 shows more than 90% amino acid similarity to the pangolin and bat CoVs and these also use ACE2 as a receptor. Binding of the spike protein to ACE2 exposes the cleavage sites to cellular proteases. Cleavage of the spike protein by transmembrane protease serine 2 and other cellular proteases initiates fusion and endocytosis. The spike protein contains an addition furin cleavage site that may allow it to be ‘preactivated’ and highly infectious after replication. The fundamental role of the spike protein in infectivity suggests that it is an important target for vaccine development, blocking therapy with antibodies and diagnostic antigen-based tests. This review briefly outlines the structure and function of the 2019 novel CoV/SARS-CoV-2 spike protein S.

scholarly journals The iminosugars celgosivir, castanospermine and UV-4 inhibit SARS-CoV-2 replication

Glycobiology ◽  
2020 ◽  
Author(s):  
Elizabeth C Clarke ◽  
Robert A Nofchissey ◽  
Chunyan Ye ◽  
Steven B Bradfute
Keyword(s):  
Viral Replication ◽  
Viral Entry ◽  
Global Economy ◽  
Viral Infections ◽  
Active Form ◽  
Viral Proteins ◽  
Spike Protein ◽  
Dependent Manner ◽  
Protein Levels ◽  
A Cell

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented challenge for health care and the global economy. Repurposing drugs that have shown promise in inhibiting other viral infections could allow for more rapid dispensation of urgently needed therapeutics. The Spike protein of SARS-CoV-2 is extensively glycosylated with 22 occupied N glycan sites and is required for viral entry. In other glycosylated viral proteins, glycosylation is required for interaction with calnexin and chaperone-mediated folding in the endoplasmic reticulum, and prevention of this interaction leads to unfolded viral proteins and thus inhibits viral replication. As such, we investigated two iminosugars, celgosivir, a prodrug of castanospermine, and UV-4, or N-(9-methoxynonyl)-1-deoxynojirimycin, a deoxynojirimycin derivative. Iminosugars are known inhibitors of the α-glucosidase I and II enzymes and were effective at inhibiting authentic SARS-CoV-2 viral replication in a cell culture system. Celgosivir prevented SARS-CoV-2-induced cell death and reduced viral replication and Spike protein levels in a dose-dependent manner in culture with Vero E6 cells. Castanospermine, the active form of celgosivir, was also able to inhibit SARS-CoV-2, confirming the canonical castanospermine mechanism of action of celgosivir. The monocyclic UV-4 also prevented SARS-CoV-2-induced death and reduced viral replication after 24 h of treatment, although the reduction in viral copies was lost after 48 h. Our findings suggest that iminosugars should be urgently investigated as potential SARS-CoV-2 inhibitors.

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