scholarly journals Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

2014 ◽  
Vol 88 (14) ◽  
pp. 7952-7961 ◽  
Author(s):  
Oliver Wicht ◽  
Wentao Li ◽  
Lione Willems ◽  
Tom J. Meuleman ◽  
Richard W. Wubbolts ◽  
...  
Keyword(s):  
Cell Culture ◽  
Fusion Protein ◽  
Natural Infection ◽  
Animal Health ◽  
Proteolytic Processing ◽  
Cell Entry ◽  
Spike Protein ◽  
Proteolytic Activation ◽  
Dependent Cell ◽  
Porcine Epidemic Diarrhea

ABSTRACTIsolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing.IMPORTANCERecurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.

TMPRSS2-Inhibitors Play a role in Cell Entry Mechanism of COVID-19: An Insight into Camostat and Nafamostat

2020 ◽  
Author(s):  
Anne Weissenstein
Keyword(s):  
Respiratory Tract ◽  
Cell Entry ◽  
Spike Protein ◽  
Cell Surfaces ◽  
Human Respiratory Tract ◽  
Proteolytic Activation ◽  
Insight Into ◽  
Entry Mechanism

The enzymes trypsin, furin and other proprotein-convertasen, cathepsin, transmembrane proteases (TMPRSS) and elastases play a role by the cell entry of Coronaviren (Coronaviridae). The proteases TMPRSS2 and TMPRSS11a, which exist in the respiratory tract richly and become experiment on cell surfaces, promote the entry of SARS-CoV-1-virus. For the TMPRSS-protease TMPRSS11d - also as protease similar to trypsin has confessed of the human respiratory tract - a proteolytic activation of the spike protein was proved by SARS-CoV.

scholarly journals The hyper-transmissible SARS-CoV-2 Omicron variant exhibits significant antigenic change, vaccine escape and a switch in cell entry mechanism

2022 ◽  
Author(s):  
Brian J Willett ◽  
Joe Grove ◽  
Oscar MacLean ◽  
Craig Wilkie ◽  
Nicola Logan ◽  
...  
Keyword(s):  
Natural Infection ◽  
Adaptive Immune Response ◽  
Cell Entry ◽  
Clinical Severity ◽  
Spike Protein ◽  
Humoral Immune ◽  
Health Strategy ◽  
Major Shift ◽  
Transmission Capability

Vaccination-based exposure to spike protein derived from early SARS-CoV-2 sequences is the key public health strategy against COVID-19. Successive waves of SARS-CoV-2 infections have been characterised by the evolution of highly mutated variants that are more transmissible and that partially evade the adaptive immune response. Omicron is the fifth of these Variants of Concern (VOCs) and is characterised by a step change in transmission capability, suggesting significant antigenic and biological change. It is characterised by 45 amino acid substitutions, including 30 changes in the spike protein relative to one of the earliest sequences, Wuhan-Hu-1, of which 15 occur in the receptor-binding domain, an area strongly associated with humoral immune evasion. In this study, we demonstrate both markedly decreased neutralisation in serology assays and real-world vaccine effectiveness in recipients of two doses of vaccine, with efficacy partially recovered by a third mRNA booster dose. We also show that immunity from natural infection (without vaccination) is more protective than two doses of vaccine but inferior to three doses. Finally, we demonstrate fundamental changes in the Omicron entry process in vitro, towards TMPRSS2-independent fusion, representing a major shift in the replication properties of SARS-CoV-2. Overall, these findings underlie rapid global transmission and may alter the clinical severity of disease associated with the Omicron variant.

scholarly journals Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by the N-terminal peptide of lactoferrin

2021 ◽  
Author(s):  
Anna Ohradanova-Repic ◽  
Laura Gebetsberger ◽  
Gabor Tajti ◽  
Gabriela Ondrovicova ◽  
Romana Prazenicova ◽  
...  
Keyword(s):  
Vero Cells ◽  
Proteolytic Processing ◽  
Cell Entry ◽  
Biologically Active ◽  
Spike Protein ◽  
Active Peptides ◽  
Biologically Active Peptides ◽  
Prevention And Treatment ◽  
Mediated Priming

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.

scholarly journals Cleavage at the Furin Consensus Sequence RAR/KR109 and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture

2002 ◽  
Vol 76 (18) ◽  
pp. 9218-9224 ◽  
Author(s):  
Gert Zimmer ◽  
Karl-Klaus Conzelmann ◽  
Georg Herrler
Keyword(s):  
Cell Culture ◽  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Reverse Genetics ◽  
Consensus Sequence ◽  
Vero Cells ◽  
Proteolytic Processing ◽  
Furin Cleavage ◽  
F Protein ◽  
Syncytial Virus

ABSTRACT Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR136) and FCS-2 (RAR/KR109). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR109, RANN109 or SANN109, respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR109 or the RANN109 motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN109 mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

scholarly journals Emergence in late 2020 of multiple lineages of SARS-CoV-2 Spike protein variants affecting amino acid position 677

2021 ◽  
Author(s):  
Emma B. Hodcroft ◽  
Daryl B. Domman ◽  
Daniel J. Snyder ◽  
Kasopefoluwa Y. Oguntuyo ◽  
Maarten Van Diest ◽  
...  
Keyword(s):  
New Mexico ◽  
Neutralizing Antibodies ◽  
Phylogenetic Analyses ◽  
Parallel Evolution ◽  
Protein S ◽  
Amino Acid Position ◽  
Proteolytic Processing ◽  
Cell Entry ◽  
Spike Protein ◽  
South Central

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) plays critical roles in host cell entry. Non-synonymous substitutions affecting S are not uncommon and have become fixed in a number of SARS-CoV-2 lineages. A subset of such mutations enable escape from neutralizing antibodies or are thought to enhance transmission through mechanisms such as increased affinity for the cell entry receptor, angiotensin-converting enzyme 2 (ACE2). Independent genomic surveillance programs based in New Mexico and Louisiana contemporaneously detected the rapid rise of numerous clade 20G (lineage B.1.2) infections carrying a Q677P substitution in S. The variant was first detected in the US on October 23, yet between 01 Dec 2020 and 19 Jan 2021 it rose to represent 27.8% and 11.3% of all SARS-CoV-2 genomes sequenced from Louisiana and New Mexico, respectively. Q677P cases have been detected predominantly in the south central and southwest United States; as of 03 Feb 2021, GISAID data show 499 viral sequences of this variant from the USA. Phylogenetic analyses revealed the independent evolution and spread of at least six distinct Q677H sub-lineages, with first collection dates ranging from mid-August to late November 2020. Four 677H clades from clade 20G (B.1.2), 20A (B.1.234), and 20B (B.1.1.220, and B.1.1.222) each contain roughly 100 or fewer sequenced cases, while a distinct pair of clade 20G clusters are represented by 754 and 298 cases, respectively. Although sampling bias and founder effects may have contributed to the rise of S:677 polymorphic variants, the proximity of this position to the polybasic cleavage site at the S1/S2 boundary are consistent with its potential functional relevance during cell entry, suggesting parallel evolution of a trait that may confer an advantage in spread or transmission. Taken together, our findings demonstrate simultaneous convergent evolution, thus providing an impetus to further evaluate S:677 polymorphisms for effects on proteolytic processing, cell tropism, and transmissibility.

scholarly journals A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: effects on proteolytic cleavage and ACE2 binding

2021 ◽  
Author(s):  
Michael G. Spelios ◽  
Jeanne M. Capanelli ◽  
Adam W. Li
Keyword(s):  
Cleavage Site ◽  
High Titer ◽  
High Sensitivity ◽  
Cell Entry ◽  
Spike Protein ◽  
Furin Cleavage ◽  
Proteolytic Activation ◽  
Blocking Antibody ◽  
Furin Cleavage Site ◽  
Site Blocking

AbstractSARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.

scholarly journals pH-Induced Conformational Change of the Rotavirus VP4 Spike: Implications for Cell Entry and Antibody Neutralization

2005 ◽  
Vol 79 (13) ◽  
pp. 8572-8580 ◽  
Author(s):  
Joseph B. Pesavento ◽  
Sue E. Crawford ◽  
Ed Roberts ◽  
Mary K. Estes ◽  
B. V. Venkataram Prasad
Keyword(s):  
Monoclonal Antibody ◽  
Sialic Acid ◽  
Conformational Change ◽  
Cell Entry ◽  
Spike Protein ◽  
Cell Binding ◽  
Ph Changes ◽  
Multiple Receptors ◽  
Neutralizing Monoclonal Antibody ◽  
Dependent Cell

ABSTRACT The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

scholarly journals Precision therapeutic targets for COVID-19

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Zachary A. Krumm ◽  
Grace M. Lloyd ◽  
Connor P. Francis ◽  
Lith H. Nasif ◽  
Duane A. Mitchell ◽  
...  
Keyword(s):  
Distress Syndrome ◽  
Replication Cycle ◽  
Spike Protein ◽  
Small Subset ◽  
High Dose ◽  
Drug Cocktail ◽  
Proteolytic Activation ◽  
Main Protease ◽  
Clinical Drugs ◽  
Length Of Hospitalization

AbstractBeginning in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 has infected more than 111 million people worldwide and caused over 2.47 million deaths. Individuals infected with SARS-CoV-2 show symptoms of fever, cough, dyspnea, and fatigue with severe cases that can develop into pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and even multi-organ failure. Current clinical management consists largely of supportive care as commonly administered treatments, including convalescent plasma, remdesivir, and high-dose glucocorticoids. These have demonstrated modest benefits in a small subset of hospitalized patients, with only dexamethasone showing demonstrable efficacy in reducing mortality and length of hospitalization. At this time, no SARS-CoV-2-specific antiviral drugs are available, although several vaccines have been approved for use in recent months. In this review, we will evaluate the efficacy of preclinical and clinical drugs that precisely target three different, essential steps of the SARS-CoV-2 replication cycle: the spike protein during entry, main protease (MPro) during proteolytic activation, and RNA-dependent RNA polymerase (RdRp) during transcription. We will assess the advantages and limitations of drugs that precisely target evolutionarily well-conserved domains, which are less likely to mutate, and therefore less likely to escape the effects of these drugs. We propose that a multi-drug cocktail targeting precise proteins, critical to the viral replication cycle, such as spike protein, MPro, and RdRp, will be the most effective strategy of inhibiting SARS-CoV-2 replication and limiting its spread in the general population.

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 Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

2006 ◽  
Vol 80 (22) ◽  
pp. 10931-10941 ◽  
Author(s):  
Rachel M. Schowalter ◽  
Stacy E. Smith ◽  
Rebecca Ellis Dutch
Keyword(s):  
Fusion Protein ◽  
Cleavage Site ◽  
Low Ph ◽  
Human Metapneumovirus ◽  
Proteolytic Processing ◽  
Luciferase Reporter ◽  
Transfected Cells ◽  
F Protein ◽  
Viral Fusion Protein ◽  
Glycosylation Sites

ABSTRACT Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

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