+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Journal of Visualized Experiments 2019(143):

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

Please choose payment method:

(PDF emailed within 1 workday: $29.90)

Accession: 066128948

Download citation: RISBibTeXText

PMID: 30688313

Related references

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proceedings of the National Academy of Sciences of the United States of America 106(14): 5871-5876, 2009

Requirement of proteolytic cleavage of the murine coronavirus MHV-2 spike protein for fusion activity. Advances in Experimental Medicine and Biology 440: 89-93, 1998

Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site. Virology 176(1): 296-301, 1990

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments. Journal of Virology 56(3): 904-911, 1985

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture. Journal of Virology 88(14): 7952-7961, 2014

Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease. Journal of Virology 85(24): 13363-13372, 2011

Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research. Antiviral Research 100(3): 605-614, 2013

Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells. Journal of Virology 83(17): 8744-8758, 2009

Acquired fusion activity of a murine coronavirus MHV-2 variant with mutations in the proteolytic cleavage site and the signal sequence of the S protein. Virology 227(1): 215-219, 1997

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion. Journal of Virology 56(3): 912-920, 1985

Comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus HKU1. Cell and Bioscience 4(1): 3, 2014

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. Journal of Clinical Microbiology 43(7): 3054-3058, 2005

Cryo-EM structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins. Plos Pathogens 14(4): E1007009, 2018

Mutation in spike protein cleavage site and pathogenesis of feline coronavirus. Emerging Infectious Diseases 19(7): 1066-1073, 2013

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity. Biochemical and Biophysical Research Communications 359(1): 174-179, 2007