Research funding for the MERS project presented in this manuscript was provided by NIH grant R21 AI111085. The feline coronavirus study was supported by research grants from Morris Animal Foundation, Winn Feline Foundation and the Cornell Feline Health Center. We also would like to thank Malik Peiris for providing us with Mor213 sequence before it was publicly accessible.

1. Designing and Preparing the Peptides
<ol>
	<li>Acquire the sequence of the fusion protein of interest from a public database such as NCBI (https://www.ncbi.nlm.nih.gov/) or the virus pathogen database (https://www.viprbrc.org). Choose the protease recognition site preceding the fusion peptide and include 2-3 amino acids upstream and downstream of this sequence.</li>
	<li>When ordering the peptides, modify them with the FRET pair 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. 
	NOTE: Several suppliers provide these modifications. Price and delivery time depend on the supplier of choice. However, alternative modifications exist which vary in sensitivity and require adjustments of the plate reader in terms of wavelengths.</li>
	<li>Resuspend the peptide according to the manufacturers recommendations by gently pipetting up and down. For example, resuspend peptides in 70% ethanol to the final concentration of 1 mM.</li>
	<li>Optionally, add the tube containing the peptide and the solvent into a sonication bath until it is fully resuspended if the peptide does not resuspend very well by pipetting.</li>
	<li>Aliquot the peptide into light dampening or resistant tubes to protect the peptide from bleaching. Keep aliquot sizes small to avoid multiple freeze/thaw cycles (e.g., 100 &#181;L). Store aliquots at -20&#176; C.</li>
</ol>
2. Preparing the Fluorescence Plate Reader
<ol>
	<li>Turn on the plate reader and wait until the self-test is finished.</li>
	<li>Open the operating software on the attached computer and make sure it is connected with the plate reader.</li>
	<li>Open the temperature setting and set to 30 &#176;C (or the required temperature for optimal performance for the protease).</li>
	<li>Click on Control | Instrument Setup to set up the experiment.</li>
	<li>Choose Kinetic.</li>
	<li>Select Fluorescence.</li>
	<li>Enter Excitation and Emission wavelengths: 330 nm and 390 nm respectively.</li>
	<li>Unselect Auto Cut-off.</li>
	<li>Select Medium, normal sensitivity.</li>
	<li>Choose a runtime of 1 h for the assay and select one measurement every 60 s.</li>
	<li>Set 5 s mixing before first measurement and 3 s before each measurement</li>
	<li>Select wells to read and start the assay.</li>
</ol>
3. Preparing the Assay
<ol>
	<li>Prepare the assay buffer by calculating the appropriate quantities for each ingredient and adding it. For furin, make buffer consisting of 100 mM HEPES, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 5% Triton X-100. For trypsin, use standard phosphate buffered saline (PBS) solution. 
	NOTE: Volumes of 10 mL or less are sufficient. Depending on the protease(s) used in the assay the buffer varies.</li>
	<li>Chill the buffer on ice.</li>
	<li>Place the assay plate on ice with a thin metal plate underneath to support cooling and stability. Use a solid black polystyrene 96-well plate with a flat bottom and non-treated. 
	NOTE: It is essential to use black plates to prevent fluorescent &#34;leakage&#34; from adjacent wells.</li>
	<li>Calculate the appropriate amount of protease to add for each reaction based on previous publications or experimental data. For furin, use 1 unit per reaction which corresponds to 0.5 &#181;L. For trypsin, use 0.5 &#181;L of 160 nM TPCK trypsin.</li>
	<li>Per sample prepare 3 technical replicates per assay in a total volume of 100 &#181;L per sample. 
	NOTE: It was experimentally evaluated that it does not make a difference whether the pipetting is carried out under normal light conditions or dimmed light. However, the peptides should not be exposed to light for an extended period of time.</li>
	<li>Pipette the appropriate amount of assay buffer (94.5 &#181;L as described under Step 3.1) into each well.</li>
	<li>Add the protease to each well. For both, furin and TPCK trypsin, use 0.5 &#181;L per sample. To 6 wells (3 wells for a blank control and 3 wells for a peptide control), add 0.5 &#181;L buffer instead of the respective protease.</li>
	<li>Add the peptide to a final concentration of 50 &#181;M to each well except the blank control. In this case, pipette 5 &#181;L peptide prepared as described under Step 1.3. Add 5 &#181;L of buffer to the blank control instead of peptide. 
	NOTE: Several concentrations of the peptide were initially tested with the described enzyme concentrations to ensure that the enzymes were saturated.</li>
	<li>Insert the plate into the fluorescence plate reader and click start.</li>
</ol>
4. Data Analysis
<ol>
	<li>Save the experiment file.</li>
	<li>Click on Export and export the file as a .txt.</li>
	<li>Import the .txt file into a spreadsheet.</li>
	<li>For each technical replicate per sample, create a graph plotting the relative fluorescent units (RFU) on the y-axis against the time on the x-axis (Supplemental Figure 1).</li>
	<li>Select the data range where the graph is in a linear range and the closest to the start of the fluorescence increase.</li>
	<li>Plot the selected data on a second graph and add a linear trendline.</li>
	<li>Select in the trendline options Display equation on chart. The equation will show the Vmax.</li>
	<li>Calculate the average Vmax for each sample from the 3 technical replicates.</li>
	<li>Repeat the experiment twice more to obtain 3 biological replicates.</li>
	<li>Calculate the standard deviation based on the data from 3 independent biological replicates.</li>
</ol>

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R., Daniel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus-mediated+membrane+fusion:+Determinants+of+hemagglutinin+fusogenic+activity+and+experimental+approaches+for+assessing+virus+fusion.">Influenza virus-mediated membrane fusion: Determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion.</a> Viruses. 4 (7), 1144-1168 (2012).</li><li>Straus, M. R., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide-based+approach+to+evaluate+the+adaptability+of+influenza+A+virus+to+humans+based+on+its+hemagglutinin+proteolytic+cleavage+site.">A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site.</a> PloS one. 12 (3), e0174827 (2017).</li><li>Kawaoka, Y., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+requirements+for+cleavage+activation+of+influenza+virus+hemagglutinin+expressed+in+mammalian+cells.">Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 85 (2), 324-328 (1988).</li><li>Abdelwhab, E. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Unique+Multibasic+Proteolytic+Cleavage+Site+and+Three+Mutations+in+the+HA2+Domain+Confer+High+Virulence+of+H7N1+Avian+Influenza+Virus+in+Chickens.">A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens.</a> Journal of Virology. 90 (1), 400-411 (2016).</li><li>Steinhauer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hemagglutinin+cleavage+for+the+pathogenicity+of+influenza+virus.">Role of hemagglutinin cleavage for the pathogenicity of influenza virus.</a> Virology. 258 (1), 1-20 (1999).</li><li>B&#246;ttcher-Friebertsh&#228;user, E., Klenk, H. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+SARS+coronavirus+spike+protein+via+sequential+proteolytic+cleavage+at+two+distinct+sites.">Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.</a> Proceedings of the National Academy of Sciences. 106 (14), 5871-5876 (2009).</li><li>Polgar, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Aspects+of+Proteases.">General Aspects of Proteases.</a> Mechanisms of Protease Action. , 43-76 (1989).</li><li>Whittaker, G. R., Andr&#233;, N. M., Millet, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+Virus+Taxonomy+by+Recontextualizing+Sequence-Based+Classification+with+Biologically+Relevant+Data:+the+Case+of+the+Alphacoronavirus+1+Species.">Improving Virus Taxonomy by Recontextualizing Sequence-Based Classification with Biologically Relevant Data: the Case of the Alphacoronavirus 1 Species.</a> mSphere. 3 (1), 1-8 (2018).</li><li>Licitra, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+in+spike+protein+cleavage+site+and+pathogenesis+of+feline+coronavirus.">Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.</a> Emerging Infectious Diseases. 19 (7), 1066-1073 (2013).</li><li>Sanjuan, R., Nebot, M. R., Chirico, N., Mansky, L. M., Belshaw, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Mutation+Rates.">Viral Mutation Rates.</a> Journal of Virology. 84 (19), 9733-9748 (2010).</li><li>Trombetta, C., Piccirella, S., Perini, D., Kistner, O., Montomoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Influenza+Strains+in+the+Last+Two+Decades:+A+Threat+of+a+New+Pandemic?.">Emerging Influenza Strains in the Last Two Decades: A Threat of a New Pandemic?.</a> Vaccines. 3 (1), 172-185 (2015).</li><li>Chu, D. K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MERS+coronaviruses+from+camels+in+Africa+exhibit+region-dependent+genetic+diversity.">MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.</a> Proceedings of the National Academy of Sciences. , 1-6 (2018).</li><li>Caprioli, R. M., Smith, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Km+and+Vmax+for+Tryptic+Peptide+Hydrolysis+Using+Fast+Atom+Bombardment+Mass+Spectrometry.">Determination of Km and Vmax for Tryptic Peptide Hydrolysis Using Fast Atom Bombardment Mass Spectrometry.</a> Analytical Chemistry. 58 (6), 1080-1083 (1986).</li><li>Millet, J. K., Goldstein, M. E., Labitt, R. N., Hsu, H., Daniel, S., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+camel-derived+MERS-CoV+with+a+variant+spike+protein+cleavage+site+and+distinct+fusion+activation+properties.">A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties.</a> Emerging microbes and infections. 5 (12), e126-e129 (2016).</li><li>Tian, S., Huajun, W., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+prediction+of+furin+cleavage+sites+by+a+hybrid+method+and+understanding+mechanism+underlying+diseases.">Computational prediction of furin cleavage sites by a hybrid method and understanding mechanism underlying diseases.</a> Scientific Reports. 2, (2012).</li><li>Hamilton, B. S., Gludish, D. W. J., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+Activation+of+the+Human-Adapted+Influenza+Virus+Subtypes+by+Matriptase+Reveals+both+Subtype+and+Strain+Specificities.">Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities.</a> Journal of Virology. 86 (19), 10579-10586 (2012).</li><li>Beaulieu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matriptase+Proteolytically+Activates+Influenza+Virus+and+Promotes+Multicycle+Epithelium+Replication+in+the+Human+Airway.">Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Epithelium Replication in the Human Airway.</a> Journal of virology. 30 (878), 4237-4251 (2013).</li><li>Hammar, L., Markarian, S., Haag, L., Lankinen, H., Salmi, A., Holland Cheng, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefusion+rearrangements+resulting+in+fusion+peptide+exposure+in+Semliki+Forest+virus.">Prefusion rearrangements resulting in fusion peptide exposure in Semliki Forest virus.</a> Journal of Biological Chemistry. 278 (9), 7189-7198 (2003).</li><li>Hamilton, B. S., Chung, C., Cyphers, S. Y., Rinaldi, V. D., Marcano, V. C., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+influenza+virus+infection+and+hemagglutinin+cleavage+by+the+protease+inhibitor+HAI-2.">Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2.</a> Biochemical and Biophysical Research Communications. 450 (2), 1070-1075 (2014).</li></ol>

The authors have nothing to disclose.

We present here a fluorogenic peptide assay that allows for a fast screening of protein sequences for their proteolytic cleavage by proteases. In our first example we chose different cleavage site motifs of the MERS-CoV spike (S) protein to illustrate one application for this assay. Several enveloped viruses that possess class I fusion proteins such as MERS-CoV, SARS-CoV (Severe Acute Respiratory Syndrome) and influenza are major concerns for public health and new subtypes are constantly evolving that have a potential to cross species barriers from their natural hosts to humans1,15,16. Isolating the viruses and cultivating them in the lab may not always be feasible or requires special laboratories that are not available in every research facility. Hence, there is a need for methods to assess the potential public health threat of emerging viruses that can be conducted under regular laboratory conditions. In addition to viruses targeting humans, some animal viruses like FCoV also possess the same class I fusion proteins, therefore, sharing the similar characteristics than their human counterparts. Isolating these viruses can also be a challenge, making the use of innovative tools to study them necessary.
A crucial step in the life cycle of the above-mentioned viruses is host cell entry and fusion mediated by proteolytic processing of the respective fusion protein by host proteases1,2. A standard way to investigate this part of the viral life cycle is to clone the fusion protein gene into a mammalian expression vector, transfect mammalian cells that allow for protein expression, incubate or co-transfect with the protease of interest, isolate the protein and perform a western blot analysis. This method comes with several limitations: availability of viral DNA/RNA for the cloning, costs for synthesizing the gene if no DNA or RNA is available, availability of an antibody to detect the fusion protein and its cleavage product(s), and it can take up to several weeks until the entire study is performed. It even becomes more time-consuming, money and labor intensive if the interest of the study is to investigate several mutations in the cleavage site because it requires site-directed mutagenesis. The fluorogenic peptide assay we describe here does not have these limitations. The peptides of interest can be designed based on publicly available sequences, there are several suppliers that provide a wide range of recombinant proteases that are relevant for these kinds of studies and the assay including the analysis can be performed within a single day.
In our example, we examined furin-mediated cleavage of the S2' protease recognition site of the MERS-CoV S protein derived from humans and camels from Egypt and Morocco. Both the human EMC/2012 and the camel HKU205 S2' sites contain a typical RXXR furin cleavage motif. However, according to the PiTou 2.0 cleavage scoring algorithm the HKU205 S2' site is not predicted to be cleaved by furin, which we experimentally confirmed19. The reason why HKU5 S2' is not processed by furin might be due to the isoleucine in the P1' position. When we tested two peptides that carried the individual mutations in the EMC/2012 S2' sequence, we were able to confirm that the isoleucine in the P1' largely abrogates cleavage whereas the alanine to serine substitution resulted in reduced cleavage. The Mor213 S2' site does not contain a furin cleavage motif and it was not surprising to detect almost no cleavage. We also examined how furin cleaves the S1/S2 and the S2' protease recognition sites of the FCoV S protein. We were able to observe furin cleavage of both FECV peptides (FECV I And-4 S1/S2 and FECV II 1683 S2'), while mutated sequences from FIPV viruses were not cleaved by furin. 
When comparing furin-mediated cleavage of the EMC/2012 S2' peptide (Figure 1A) with the FECV I And-4 S1/2 peptides (Figure 2A), we found that there was an approximate 26-fold difference in the Vmax. This can be explained by the furin cleavage motif in both sequences (Table 1). While the minimum requirement for furin cleavage is a RXXR motif, a RXRR sequence is much more favorable2. Therefore, the EMC/2012 S2' peptide, which has a RSAR motif, is cleaved with less specificity compared to the FECV I And-4 S1/2 peptide that contains a RSRR furin cleavage site (Table 1). 
However, one major limitation of the assay is that the peptides do not reflect the tertiary structure of the protein they are usually embedded in. Cleavage of the full length fusion protein exposes the fusion peptide and triggers host cell entry1. The interaction between protease and fusion protein might also be impacted by the tertiary structure of the fusion protein while we assume that the peptides are more or less linear. Hence, cleavage detected in the peptide assay may be artificial and may not reflect the in vivo situation. This was reported for cleavage of the influenza H3N2 HA subtype by matriptase. While several groups described cleavage of the H3N2 HA by matriptase in peptide assays, it was also shown that incubation of full length H3N2 HA protein and matriptase in cell culture did not result in a fusogenic HA protein4,20,21. There are other examples that show that the biologically important regulation of the proteolytic cleavage is on the level of protein conformation. It has been described that fusion proteins sometimes need a series of pre-fusion events to be able to expose the cleavage sites upon fusion. The Semliki Forest virus fusion protein, for example, requires structural rearrangements during a number of prefusion events in order to expose its fusion protein and make it available for proteolytic activation22. Therefore, the results obtained in a fluorogenic peptide assay need to be validated by cell fusion assays or similar experiments. However, the peptide assay still allows a fast screening of several protease/peptide combinations and to reduce the labor and time of follow up experiments since combinations that do not show cleavage are very likely to exhibit the same result in vivo.
The second major limitation of the assay concerns the proteases. So far it is only possible to test soluble proteases but not transmembrane proteases. Depending on the intent of the experiment, this can be a problem. For example, influenza HAs are activated by a number of transmembrane proteases located at cell membranes8. To a certain extent, this problem can be circumvented by using the soluble proteolytic active domains, but the results have to be interpreted with caution and should be validated with conventional methods. We want to refer to the beforementioned example with matriptase and its activity towards H3N2 HA. In vivo matriptase is located in the cell membrane but the commercially available enzyme is the soluble catalytic domain. As discussed with respect to the fusion proteins, it may be possible that it also requires the full-length protease to interact with the full-length protein substrate to obtain cleavage and that testing only single domains may result in false positives. 
This methodology has shown to be efficient to study the cleavage of specific amino acid sequences by furin. However, the applications of the technique extents to any available protease (e.g., cathepsins, proprotein convertase), making it a useful method to screen peptide candidates for protease cleavage. Moreover, it has been shown that this assay can also be used to test the efficacy of protease inhibitors and hence provides a fast and easy to tool to screen protein inhibitors23. 
The fluorogenic peptide cleavage assay described here represents an addition to bioinformatic cleavage scoring algorithms, which predicts the peptide cleavage by a determined protease, based on the amino acid properties and sequence. These two techniques, in combination with tradition western blotting techniques can be used as an efficient method to study protease cleavage in vitro.

Viral fusion with the host cell membrane is a crucial step in the life cycle of enveloped viruses and facilitates entry into the cell and replication of the virus. Typically, viruses possess a specialized protein (or proteins) that binds to receptors on the host cell membrane and triggers the virus-cell membrane fusion1. Viral fusion proteins have been grouped into three main classes (I-III)1. A number of viruses that are currently a major concern for public health, such as influenza virus (representing a long-standing zoonotic emergence from birds) and Middle East Respiratory Syndrome-coronavirus (MERS-CoV) (representing a recent zoonotic emergence from camels), utilize the so-called class I fusion proteins, which require proteolytic processing by host proteases, to exert their fusogenic activity2. Similarly, feline coronavirus (FCoV), which represents a major disease threat for wild and domestic cats, also possess a class I fusion protein. Class I viral fusion proteins are typically synthesized as an uncleaved precursor, and generally consist of two domains that are responsible for receptor binding and triggering the fusion event respectively. To date, the influenza hemagglutinin (HA) is the best understood fusion protein and numerous studies have described its mechanistic role in host cell entry and fusion3. In this case cleavage of the fusion protein occurs at a specific sequence or cleavage site within HA, and in combination with pH-dependent conformational changes, results in the exposure of the viral fusion peptide1,2.
The fusion peptide and its preceding protease recognition sequence are critical for the pathogenicity and the host adaptation of the virus. Changes in the protease recognition sequence can alter cleavage significantly with potentially dramatic consequences for the virus and the host4. On one hand, it can abrogate cleavage and thus eliminate the life cycle of the virus. But on the other hand, a mutation can increase the substrate specificity for a given protease and/or allow a &#34;new&#34; protease to cleave the fusion protein. Subsequently, this can also expand the cell and tissue tropism as observed, e.g., with influenza virus subtypes5,6. Usually low pathogenicity avian influenza (LPAI) viruses and most human influenza viruses are confined to the gastrointestinal or respiratory tract because of the limited localization of the proteases that activate them. Typically, the fusion peptide of such HA proteins is preceded by a four-amino acid sequence that consists of 1-2 non-consecutive basic amino acids, which is recognized by trypsin-like serine proteases like trypsin, matriptase or TMPRSS27,8. When the virus acquires insertions or mutations that change the cleavage site to a polybasic site, it allows furin to activate the HA and to potentially cause a much more severe systemic infection6,9. These viruses are referred to as high pathogenicity avian influenza virus (HPAI), typified by H5N1 strains.
In contrast to influenza HA, many coronaviruses, such as MERS-CoV, have two distinct cleavage sites within their spike protein. The S1/S2 site separates the N-terminal receptor binding domain (S1) from the C-terminal fusion domain (S2), with a second cleavage site, called S2&#39;, downstream of the S1/S2 site, in proximity to the fusion peptide10. It was suggested that the sites are cleaved sequentially, at S1/S2 followed by S2&#39;. In contrast to the HA of most influenza virus strains, the MERS-CoV S protein S1/S2 and S2&#39; sites can be recognized by proteases of the proprotein convertase (PC) family, such as furin. Members of this family cleave at paired basic residues with the motif R/K-(X)0,2,4,6-R/K(X, any amino acid)2. Generally, the amino acids upstream of the cleavage site are referred to as P1, P2, P3, etc. counting from the cleavage site and the amino acids downstream are designated as P1&#39;, P2&#39;, P3&#39;, etc.11. FCoV strains can either have a single or a dual cleavage site. Like MERS-CoV, some FCoV strains also possess two cleavage sites (S1/S2 and S2&#39;) in their S protein. However, this characteristic is exclusive of serotype I FCoVs (clade A). In contrast, members of the serotype II (clade B) grouping only have a single cleavage site (S2&#39;)2,12. Several proteases have been suggested to cleave the FCoV cleavage sites, including furin, trypsin-like proteases and cathepsin. It has been proposed that the S protein of enteric FCoV (also known as feline enteric coronavirus or FECV) is likely to be cleaved by furin in the S1/S2 site, and mutations in this site (as well as at S2&#39;) leads to changes in protease requirements. These mutations have been associated with changes in the tropism and pathogenicity of these viruses, allowing the virus to become systemic and macrophage-tropic (also known as feline infectious peritonitis virus or FIPV)13.
Viruses naturally introduce mutations into their genomes during each replication cycle and frequently new subtypes and strains of&#160;influenza, MERS-CoV and FCoV are described14,15,16. As a part of a rapid evaluation to assess the public health threat of emerging viruses, it is critical to investigate changes in the cleavage site and how it affects the range of proteases activating these viruses. Here, we describe a peptide-based assay that allows a very quick assessment of how the cleavage site changes in MERS-CoV S protein affect the substrate specificity of a given protease and to rapidly screen various proteases for their ability to cleave a given or multiple sequences4. In a second set of experiments, we used the technique to determine the furin cleavage activity over different FCoV serotypes and strains.
The peptides used in this assay are modified with the fluorescence resonance energy transfer (FRET) pair, 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. During the assay, MCA is excited and emits light energy that is quenched by DNP as long as the pair is in close vicinity to each other. If cleavage occurs, however, DNP is not able to quench the emission anymore and it can be read by the fluorescence plate reader. The changes in the fluorescence is measured during the experiment to determine the peptide cleavage rate, and to calculate the velocity at which the protease cleaves the specific peptide (also known as Vmax)17.
In order to design the peptides, the gene of the respective fusion protein must have been sequenced and/or made available in a database. However, the method is less labor-intense and costly than conventional methods that usually require the cloning of the fusion protein gene into expression vectors to express it in mammalian cells to analyze the cleavage. From start to end, this may take several days up to a few weeks while the peptide assays presented here can be done within one day as soon as peptides and proteases are available. The setup of the assay takes between 5 and 30 min depending on the number of samples and the runtime in the fluorescence plate reader is 1 h. Analysis of the data may take up to 2 h again depending on the sample size.
Here, we chose two different examples to present the assay. In the first example, we present data that compares the furin-mediated cleavage of human and camel-derived MERS-CoV, to assess the potential of the camel-derived strains of being activated in humans if they cross the species barrier. In the second example, we used a fluorogenic peptide assay to determine the furin-mediated cleavage of the S1/S2 and S2&#39; sites or S2&#39; site of two serotype I and two serotype II FCoV strains, respectively. For these experiments, we used trypsin cleavage as a positive control.

<table><tbody><tr><td>Peptides</td><td>Biomatik</td><td>N/A</td><td></td></tr><tr><td>Furin</td><td>NEB</td><td>P8077S</td><td></td></tr><tr><td>Trypsin, TPCK-treated</td><td>Sigma-Aldrich</td><td>4352157-1KT</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>SpectraMax Gemini XPS</td><td>Molecular Devices</td><td>XPS</td><td></td></tr><tr><td>SoftMax Pro 6.5.1&amp;nbsp;</td><td>Molecular Devices</td><td>N/A</td><td></td></tr><tr><td>96-well plate (solid black polystyrene with a flat bottom and non-treated)</td><td>Costar</td><td>3915</td><td></td></tr><tr><td>Light damping tubes</td><td>Watson Lab</td><td>131-915BL or 131-915BR</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>N/A</td><td></td></tr><tr><td>Prism 7</td><td>GraphPad</td><td>N/A</td><td></td></tr></tbody></table>

a fluorogenic peptide cleavage assay to screen for proteolytic activity: applications for coronavirus spike protein activation

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.

In the first part of this study, we used peptides that represent the S2&#39; cleavage site of three distinct MERS-CoV S proteins: from the prototypical EMC/2012 human strain (Genbank AFS88936.1) and from two selected camel-derived MERS-CoVs, NRCE-HKU205 (Genbank AHL18090.1) and Mor215 (Genbank AVN89324.1) strains, isolated in Egypt and Morocco, respectively (Table 1)16,18.
Compared to the EMC/2012 strain, the HKU205 S2&#39; cleavage site has two mutations in the P2 and P1&#39; positions that could potentially impact its recognition by furin and alter the cleavage efficiency (Table 1). In addition, we were interested to investigate if and how each individual mutation found in the HKU205 strain affects furin cleavage. We, therefore, included two peptides where the individual substitutions were introduced into the EMC/2012 peptide sequence (EMC/2012 mutA-S S2&#39; and EMC/2012 mutS-I S2&#39;) (Table 1). Furin was able to cleave the EMC/2012 peptide efficiently with a Vmax of 6.3 &#177; 1.2 RFU/min, while we detected almost no cleavage when using the S2&#39; peptide of the HKU205 strain (Vmax 0.5 &#177; 0.1 RFU/min) (Figure 1a). When investigating the EMC/2012 mutA-S S2&#39; peptide, we found that the cleavage was strongly reduced compared to the wild type sequence (Vmax 3.6 &#177; 1 RFU/min). There was almost no cleavage when testing the EMC/2012 mutS-I S2&#39; peptide (Vmax 1.1 &#177; 0.9 RFU/min) (Figure 1a).
Recently, MERS-CoV isolates from Western Africa were reported that are phylogenetically distinct from MERS-CoV found in the Arabian Peninsula16. One of the recently described isolates is the Mor213 strain, which carries a leucine instead of an arginine in the P4 position of the S2&#39; site (Table 1), and which could potentially impact its recognition by furin and alter the cleavage efficiency. Our peptide assay showed that there is only minimal cleavage of the Mor213 S2&#39; site as compared to the EMC/2012 site (Figure 1b). The Vmax for the Mor213 peptide is 1.0 &#177; 0.1.
For the second part of this study, we used the same peptide assay to evaluate furin-mediated cleavage of FCoV S protein cleavage sites. We used peptides mimicking the S1/S2 cleavage site of two FCoV serotype I viruses: FECV I And-4 (from Whittaker lab) and FIPV I Black (Genbank AB088223.1). We also used peptides mimicking the S2&#39; site of these viruses, as well as two FCoV serotype II: FECV II 1683 (Genbank AFH58021.1) and FIPV II 1146 (Genbank AAY32596.1) strains (Table 1). Typically, furin cleavage occurs when basic residues (arginine and lysine) are present in the P1 and P4 positions of the cleavage site. Mutations in the P1, P4 and P1&#39; positions are suggested to interfere with furin cleavability, therefore altering the protease requirements of the protein. (Table 1). Furin cleavage was observed for the prototypical S1/S2 peptide FECV I And-4 S1/S2 (Vmax 100.36 &#177; 7.61 RFU/min) but not in the mutated S1/S2 peptide FIPV I Black S1/S2 (Vmax -1.37 &#177; 1.29 RFU/min) (Figure 2A). Similarly, furin was able to cleave the prototypical S2&#39; peptide FECV II 1683 S2&#39; (Vmax 5.46 &#177; 0.97 RFU/min), but not the mutated S2&#39; peptides FECV I And-4 S2&#39; (Vmax 0.26 &#177; 0.11 RFU/min), FIPV I Black S2&#39; (Vmax 0.30 &#177; 0.14 RFU/min) and FIPV II 1146 S2&#39; (Vmax -0.36 &#177; 0.11 RFU/min) (Figure 2B). We used trypsin as a positive control for peptide cleavage and we observed trypsin cleavage in all the peptides used (Supplemental Figure 2).
<img alt="Table 1" src="/files/ftp_upload/58892/58892tbl1v2.jpg" />
Table 1. Peptides used and corresponding amino acid sequences. The peptides used in the assays contain the (7-methoxycoumarin-4-yl) acetyl/2,4-dinitrophenyl (MCA/DNP) FRET pair. The P4 and P1 cleavage site positioned arginine (R) residues, which are recognized by furin, are in bold. Residues in red correspond to mutations compared to reference sequences.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58892/58892fig1v2.jpg" /> 
Figure 1. Furin-mediated proteolytic cleavage of human and camel-derived MERS-CoV S2&#39; sites fluorogenic peptides. A. Furin cleavage assay of human MERS-CoV EMC/2012 S2&#39; site and camel-derived strain HKU205 (double mutant), along with single mutant variants of EMC/2012 (mutA-S and mutS-I). B. Furin cleavage assay of human MERS-CoV EMC/2012 S2&#39; site and camel-derived strain Mor213. For panels A and B, peptides were incubated with recombinant furin and the increase in fluorescence due to proteolytic processing was measured using a fluorometer. The assays were performed in triplicates with results representing averages of Vmax from three independent experiments (n=3). Error bars indicate SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58892/58892fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58892/58892fig2.jpg" /> 
Figure 2. Furin-mediated proteolytic cleavage of FCoV S1/S2 and S2&#39; sites fluorogenic peptides.&#160;A. Furin cleavage assay of FECV I And-4 and FIPV I Black S1/S2 sites. Peptides were incubated with recombinant furin. B. Furin cleavage assay of FECV I And-4, FIPV I Black, FECV II 1683 and FIPV II 1146 S2&#39; sites. The increase in fluorescence due to proteolytic processing was measured using a fluorometer. The assays were performed in triplicates with results representing averages of Vmax from three independent experiments (n=3). Error bars indicate SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58892/58892fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table 1: Vmax values used for Figure 1 and Figure 2. The Vmax values were calculated from graphs as illustrated in Supplemental Figure 1 and as described in Protocol section 4.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58892/Supplemental_Table_1.xlsx" target="_blank">Please click here to download this table.</a>
Supplemental Figure 1: Illustration of raw data derived from the plate reader software. Increase of fluorescence over time graphs that were used for determination of the Vmax.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58892/Fig_S1.ai" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Trypsin-mediated proteolytic cleavage of FCoV S1/S2 and S2&#39; sites fluorogenic peptides. Trypsin cleavage assay of FECV I And-4 and FIPV I Black S1/S2 sites and of FECV I And-4, FIPV I Black, FECV II 1683 and FIPV II 1146 S2&#39; sites.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58892/Supplemental_Figure_2.eps" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation at JoVE.com

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

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

a-fluorogenic-peptide-cleavage-assay-to-screen-for-proteolytic

Nanyang Technological University

Research

JoVE Journal

Biochemistry

33.1K Views.  Cornell University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van biochemie, zoals de proteolytische modificatie van eiwitten, evenals de karakterisering van remmers en proteases en peptidases. Het belangrijkste voordeel van deze techniek is dat het een snelle screening van de proteolytische activiteit van proteasen op peptiden mogelijk maakt, die de decolletékant van het gegeven eiwit vertegenwoordigen. Het gebruik hier is om proteases te bestuderen die virale fusie acti...

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

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cancer Research

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Biology

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Bioengineering

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Immunology and Infection

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce ...

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

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

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