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Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.

Licitra BN, Millet JK, Regan AD, Hamilton BS, Rinaldi VD, Duhamel GE, Whittaker GR - Emerging Infect. Dis. (2013)

Bottom Line: FECV sequences were compared with FIPV sequences.Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage.These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

View Article: PubMed Central - PubMed

Affiliation: Cornell University College of Veterinary Medicine,Ithaca, New York 14853, USA.

ABSTRACT
Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

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Furin cleavage assays of fluorogenic peptides. A) Synthetic fluorogenic peptides were generated with sequences matching consensus feline enteric coronavirus and a panel of modified sequences with substitutions (shown in red) found by feline infectious peritonitis virus sequencing. Peptides (50 μmol/L) were subjected to cleavage by recombinant human furin (1 U/100 μL), at pH 7.5, 30°C, and the release of fluorescence over time was measured by a spectrofluorometer enabling calculation of the Vmax of each reaction. Peptide cleavage scores generated by the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) are also displayed. B) For each modified peptide (substitutions shown in red), the percentage of cleavage rate compared with the canonical sequence was calculated and displayed. Cleavage assays were performed in >3 independent experiments. Error bars indicate SD for each measurement.
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Figure 4: Furin cleavage assays of fluorogenic peptides. A) Synthetic fluorogenic peptides were generated with sequences matching consensus feline enteric coronavirus and a panel of modified sequences with substitutions (shown in red) found by feline infectious peritonitis virus sequencing. Peptides (50 μmol/L) were subjected to cleavage by recombinant human furin (1 U/100 μL), at pH 7.5, 30°C, and the release of fluorescence over time was measured by a spectrofluorometer enabling calculation of the Vmax of each reaction. Peptide cleavage scores generated by the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) are also displayed. B) For each modified peptide (substitutions shown in red), the percentage of cleavage rate compared with the canonical sequence was calculated and displayed. Cleavage assays were performed in >3 independent experiments. Error bars indicate SD for each measurement.

Mentions: To test whether the identified FIPV S1/S2 mutations have an effect on cleavability by furin, we performed an in vitro proteolytic assay. We used human furin for these experiments. Human and feline furin are very similar (96% identical) and are expected to cleave in an equivalent manner. However, feline furin has not been directly studied to any degree, and reagents are not readily available. Feline and human cells lines show identical rates of cleavage for a known furin target protein (PSCK-9), which contains an active furin cleavage site (Technical Appendix Figure 2). We used fluorogenic peptides containing the canonical motif (R-R-S-R-R-S) or with substitutions from positions P1′ through P7 (Figure 4, panel A). The canonical peptide was efficiently cleaved by furin (Figure 4), with average Vmax of 235 Relative Fluorescence Units (RFU) per minute.


Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.

Licitra BN, Millet JK, Regan AD, Hamilton BS, Rinaldi VD, Duhamel GE, Whittaker GR - Emerging Infect. Dis. (2013)

Furin cleavage assays of fluorogenic peptides. A) Synthetic fluorogenic peptides were generated with sequences matching consensus feline enteric coronavirus and a panel of modified sequences with substitutions (shown in red) found by feline infectious peritonitis virus sequencing. Peptides (50 μmol/L) were subjected to cleavage by recombinant human furin (1 U/100 μL), at pH 7.5, 30°C, and the release of fluorescence over time was measured by a spectrofluorometer enabling calculation of the Vmax of each reaction. Peptide cleavage scores generated by the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) are also displayed. B) For each modified peptide (substitutions shown in red), the percentage of cleavage rate compared with the canonical sequence was calculated and displayed. Cleavage assays were performed in >3 independent experiments. Error bars indicate SD for each measurement.
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Related In: Results  -  Collection

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Figure 4: Furin cleavage assays of fluorogenic peptides. A) Synthetic fluorogenic peptides were generated with sequences matching consensus feline enteric coronavirus and a panel of modified sequences with substitutions (shown in red) found by feline infectious peritonitis virus sequencing. Peptides (50 μmol/L) were subjected to cleavage by recombinant human furin (1 U/100 μL), at pH 7.5, 30°C, and the release of fluorescence over time was measured by a spectrofluorometer enabling calculation of the Vmax of each reaction. Peptide cleavage scores generated by the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) are also displayed. B) For each modified peptide (substitutions shown in red), the percentage of cleavage rate compared with the canonical sequence was calculated and displayed. Cleavage assays were performed in >3 independent experiments. Error bars indicate SD for each measurement.
Mentions: To test whether the identified FIPV S1/S2 mutations have an effect on cleavability by furin, we performed an in vitro proteolytic assay. We used human furin for these experiments. Human and feline furin are very similar (96% identical) and are expected to cleave in an equivalent manner. However, feline furin has not been directly studied to any degree, and reagents are not readily available. Feline and human cells lines show identical rates of cleavage for a known furin target protein (PSCK-9), which contains an active furin cleavage site (Technical Appendix Figure 2). We used fluorogenic peptides containing the canonical motif (R-R-S-R-R-S) or with substitutions from positions P1′ through P7 (Figure 4, panel A). The canonical peptide was efficiently cleaved by furin (Figure 4), with average Vmax of 235 Relative Fluorescence Units (RFU) per minute.

Bottom Line: FECV sequences were compared with FIPV sequences.Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage.These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

View Article: PubMed Central - PubMed

Affiliation: Cornell University College of Veterinary Medicine,Ithaca, New York 14853, USA.

ABSTRACT
Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Show MeSH
Related in: MedlinePlus