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

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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SARS-CoV-2 Origin: An Affair of Codons?

10.20944/preprints202106.0121.v1 ◽

2021 ◽

Author(s):

Antonio Ramón Romeu ◽

Enric Ollé

Keyword(s):

Human Genome ◽

Genetic Markers ◽

Cleavage Site ◽

Cleavage Sites ◽

Furin Cleavage ◽

Large Sample ◽

The Core ◽

Furin Cleavage Site

The furin cleavage site, with an arginine doublet (RR), is one of the clues of the SARS-CoV-2 origin. This furin-RR is encoded by the CGG-CGG sequence. Because arginine can be encoded by six codons, in a previous work we found that in SARS-CoV-2, CGG was the minority arginine codon (3%). Also, analyzing the RR doublet from a large sample of furin cleavage sites of several kinds of viruses, we found that none of them were encoded by CGG-CGG. Here, we come back to the core of the matter, but from the perspective that in the human genome, in contrast, CGG is the majoroty arginine codon (21%). Here, we highlighted that the 6 arginine codons provide genetic markers to a traceability on the RR origin in the furin site, as well as, to weigh the probability of the theories about the origin of the virus.

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The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia

10.1101/2021.02.03.429670 ◽

2021 ◽

Author(s):

Wei Zou ◽

Min Xiong ◽

Siyuan Hao ◽

Elizabeth Yan Zhang-Chen ◽

Nathalie Baumlin ◽

...

Keyword(s):

Amino Acids ◽

Viral Genome ◽

Cleavage Site ◽

Vero Cells ◽

Furin Cleavage ◽

Airway Epithelia ◽

Human Airway ◽

S Gene ◽

Furin Cleavage Site ◽

Human Airway Epithelia

AbstractThe novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682RRAR↓S686, distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678TNSPRRAR↓SVAS689, which contains the FCS, another is a deletion of 5 amino acids, 675QTQTN679, which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is donor-dependent.

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Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109905118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2109905118

Author(s):

Liping Zhang ◽

Matthew Mann ◽

Zulfeqhar A. Syed ◽

Hayley M. Reynolds ◽

E. Tian ◽

...

Keyword(s):

Cleavage Site ◽

Protein S ◽

Viral Infectivity ◽

The Novel ◽

Furin Cleavage ◽

Global Pandemic ◽

Furin Cleavage Site ◽

Respiratory Cells ◽

Syncytia Formation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

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Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Journal of Virology ◽

10.1128/jvi.78.24.13573-13581.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13573-13581 ◽

Cited By ~ 25

Author(s):

Verena Geiselhart ◽

Patrizia Bastone ◽

Tore Kempf ◽

Martina Schnölzer ◽

Martin Löchelt

Keyword(s):

Cleavage Site ◽

Transmembrane Protein ◽

Foamy Virus ◽

Proteolytic Processing ◽

Signal Peptidase ◽

Amino Acid Residues ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Leader Protein ◽

Distinguishing Features

ABSTRACT The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.

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Abstract 095: Mutation in the Furin Cleavage Site of the Prorenin Receptor Attenuates Angiotensin-II Induced Hypertension and Albuminuria

Hypertension ◽

10.1161/hyp.74.suppl_1.095 ◽

2019 ◽

Vol 74 (Suppl_1) ◽

Author(s):

Nirupama Ramkumar ◽

Deborah Stuart ◽

Will Wheatley ◽

Donald E Kohan

Keyword(s):

Angiotensin Ii ◽

Cleavage Site ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Prorenin Receptor ◽

Induced Hypertension

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TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

AJP Renal Physiology ◽

10.1152/ajprenal.00330.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. F1-F8 ◽

Cited By ~ 30

Author(s):

Christopher J. Passero ◽

Gunhild M. Mueller ◽

Michael M. Myerburg ◽

Marcelo D. Carattino ◽

Rebecca P. Hughey ◽

...

Keyword(s):

Sodium Channel ◽

Cleavage Site ◽

Epithelial Sodium Channel ◽

Cleavage Sites ◽

Furin Cleavage ◽

Channel Activation ◽

Α Subunit ◽

Γ Subunit ◽

Furin Cleavage Site ◽

Unique Mechanism

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.

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Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

Journal of Virology ◽

10.1128/jvi.02304-20 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hanh T. Nguyen ◽

Shijian Zhang ◽

Qian Wang ◽

Saumya Anang ◽

Jia Wang ◽

...

Keyword(s):

Host Cell ◽

Cell Fusion ◽

Cleavage Site ◽

Virus Entry ◽

Cytoplasmic Tail ◽

Syncytium Formation ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

Cytopathic Effects ◽

Furin Cleavage Site

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

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