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Signal peptide cleavage from GP5 of PRRSV: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence.

Thaa B, Sinhadri BC, Tielesch C, Krause E, Veit M - PLoS ONE (2013)

Bottom Line: This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains.The results revealed that the signal peptide of GP5 is cleaved at two sites.This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Department of Veterinary Medicine, Free University Berlin, Berlin, Germany.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a "decoy epitope" located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the "decoy epitope" is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the "decoy epitope" sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".

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Identification of the signal peptide cleavage site of GP5 (virus-derived and recombinant) by mass spectrometry.(A), PRRSV (strain VR-2332) was grown in MARC-145 cells, precipitated with PEG-8000, pelleted and subjected to sucrose density gradient centrifugation. The virus-containing fraction was left untreated or deglycosylated with PNGase F and separated by reducing SDS-PAGE followed by Coomassie staining (left-hand side) or Western blot (anti-GP5 antiserum, right-hand side). The deglycosylated band corresponding to GP5 (black box) was cut out of the gel, digested with trypsin or chymotrypsin and analyzed by LC-MS/MS. (B), PRRSV GP5 (with His tag) and M (with HA tag) were co-expressed in Sf9 insect cells by infection with recombinant baculovirus. Following cell harvesting and lysis, GP5–M was enriched using Ni-NTA agarose (binding to GP5–His). The eluated protein was left untreated or digested with PNGase F and subjected to reducing SDS-PAGE and Coomassie staining (left-hand side) or Western blot (anti-His-tag antibody, right-hand side). The deglycosylated GP5 band (black box; coinciding with M) was cut out and treated as in (A). (C), representative result from mass spectrometry of virus-derived GP5 (as in A). The first 61 residues of GP5 are shown with the positions of the predicted chymotrypsin cleavage sites (black lines) and the putative signal peptide cleavage sites (broken lines). Chymotryptic peptides that were identified are represented as black bars. The pattern of peptides is evidence for signal peptide cleavage at sites 1 and 2. No peptides corresponding to the signal peptide region (1–26) were identified. (D), Conclusion from mass spectrometry, showing the N-terminal sequence of GP5 with signal peptide (black), glycosylations (grey) and the positions of the neutralizing and the “decoy epitope”. Two GP5 species exist with signal peptide cleavage at sites 2 (top) and 1 (bottom), respectively.
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pone-0065548-g007: Identification of the signal peptide cleavage site of GP5 (virus-derived and recombinant) by mass spectrometry.(A), PRRSV (strain VR-2332) was grown in MARC-145 cells, precipitated with PEG-8000, pelleted and subjected to sucrose density gradient centrifugation. The virus-containing fraction was left untreated or deglycosylated with PNGase F and separated by reducing SDS-PAGE followed by Coomassie staining (left-hand side) or Western blot (anti-GP5 antiserum, right-hand side). The deglycosylated band corresponding to GP5 (black box) was cut out of the gel, digested with trypsin or chymotrypsin and analyzed by LC-MS/MS. (B), PRRSV GP5 (with His tag) and M (with HA tag) were co-expressed in Sf9 insect cells by infection with recombinant baculovirus. Following cell harvesting and lysis, GP5–M was enriched using Ni-NTA agarose (binding to GP5–His). The eluated protein was left untreated or digested with PNGase F and subjected to reducing SDS-PAGE and Coomassie staining (left-hand side) or Western blot (anti-His-tag antibody, right-hand side). The deglycosylated GP5 band (black box; coinciding with M) was cut out and treated as in (A). (C), representative result from mass spectrometry of virus-derived GP5 (as in A). The first 61 residues of GP5 are shown with the positions of the predicted chymotrypsin cleavage sites (black lines) and the putative signal peptide cleavage sites (broken lines). Chymotryptic peptides that were identified are represented as black bars. The pattern of peptides is evidence for signal peptide cleavage at sites 1 and 2. No peptides corresponding to the signal peptide region (1–26) were identified. (D), Conclusion from mass spectrometry, showing the N-terminal sequence of GP5 with signal peptide (black), glycosylations (grey) and the positions of the neutralizing and the “decoy epitope”. Two GP5 species exist with signal peptide cleavage at sites 2 (top) and 1 (bottom), respectively.

Mentions: The observed migration pattern of deglycosylated GP5–HA is clear evidence for signal peptide cleavage; yet, the exact site of signal peptide cleavage cannot be derived from these data. We therefore employed mass spectrometry as a powerful tool to unequivocally identify proteins and protein fragments. Immunoprecipitation of cell-expressed GP5–HA does not yield enough material for mass spectrometry (data not shown). Therefore, we grew large quantities of PRRSV, strain VR-2332, in MARC-145 cells and partially purified the virus by PEG-8000 precipitation, ultracentrifugation and sucrose density gradient centrifugation (Fig. 7A). Virus proteins were subjected to PNGase F digestion to remove glycans and were separated by SDS-PAGE and stained with Coomassie. Deglycosylation is necessary to avoid heterogeneity in peptides due to the presence of differentially processed carbohydrate side chains. In addition, as a result of deglycosylation with PNGase F, asparagine is converted into aspartic acid and thus the occurrence of D instead of N is evidence that a certain glycosylation site is actually used. The band corresponding to GP5 (running at 16 kDa only after PNGase F digestion) was excised from the gel (see Fig. 7A). The band was verified to be GP5 by Western blot run in parallel, yielding a signal for GP5 at the same height as the excised band (Fig. 7A, right-hand panel). Two independent preparations were carried out.


Signal peptide cleavage from GP5 of PRRSV: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence.

Thaa B, Sinhadri BC, Tielesch C, Krause E, Veit M - PLoS ONE (2013)

Identification of the signal peptide cleavage site of GP5 (virus-derived and recombinant) by mass spectrometry.(A), PRRSV (strain VR-2332) was grown in MARC-145 cells, precipitated with PEG-8000, pelleted and subjected to sucrose density gradient centrifugation. The virus-containing fraction was left untreated or deglycosylated with PNGase F and separated by reducing SDS-PAGE followed by Coomassie staining (left-hand side) or Western blot (anti-GP5 antiserum, right-hand side). The deglycosylated band corresponding to GP5 (black box) was cut out of the gel, digested with trypsin or chymotrypsin and analyzed by LC-MS/MS. (B), PRRSV GP5 (with His tag) and M (with HA tag) were co-expressed in Sf9 insect cells by infection with recombinant baculovirus. Following cell harvesting and lysis, GP5–M was enriched using Ni-NTA agarose (binding to GP5–His). The eluated protein was left untreated or digested with PNGase F and subjected to reducing SDS-PAGE and Coomassie staining (left-hand side) or Western blot (anti-His-tag antibody, right-hand side). The deglycosylated GP5 band (black box; coinciding with M) was cut out and treated as in (A). (C), representative result from mass spectrometry of virus-derived GP5 (as in A). The first 61 residues of GP5 are shown with the positions of the predicted chymotrypsin cleavage sites (black lines) and the putative signal peptide cleavage sites (broken lines). Chymotryptic peptides that were identified are represented as black bars. The pattern of peptides is evidence for signal peptide cleavage at sites 1 and 2. No peptides corresponding to the signal peptide region (1–26) were identified. (D), Conclusion from mass spectrometry, showing the N-terminal sequence of GP5 with signal peptide (black), glycosylations (grey) and the positions of the neutralizing and the “decoy epitope”. Two GP5 species exist with signal peptide cleavage at sites 2 (top) and 1 (bottom), respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675037&req=5

pone-0065548-g007: Identification of the signal peptide cleavage site of GP5 (virus-derived and recombinant) by mass spectrometry.(A), PRRSV (strain VR-2332) was grown in MARC-145 cells, precipitated with PEG-8000, pelleted and subjected to sucrose density gradient centrifugation. The virus-containing fraction was left untreated or deglycosylated with PNGase F and separated by reducing SDS-PAGE followed by Coomassie staining (left-hand side) or Western blot (anti-GP5 antiserum, right-hand side). The deglycosylated band corresponding to GP5 (black box) was cut out of the gel, digested with trypsin or chymotrypsin and analyzed by LC-MS/MS. (B), PRRSV GP5 (with His tag) and M (with HA tag) were co-expressed in Sf9 insect cells by infection with recombinant baculovirus. Following cell harvesting and lysis, GP5–M was enriched using Ni-NTA agarose (binding to GP5–His). The eluated protein was left untreated or digested with PNGase F and subjected to reducing SDS-PAGE and Coomassie staining (left-hand side) or Western blot (anti-His-tag antibody, right-hand side). The deglycosylated GP5 band (black box; coinciding with M) was cut out and treated as in (A). (C), representative result from mass spectrometry of virus-derived GP5 (as in A). The first 61 residues of GP5 are shown with the positions of the predicted chymotrypsin cleavage sites (black lines) and the putative signal peptide cleavage sites (broken lines). Chymotryptic peptides that were identified are represented as black bars. The pattern of peptides is evidence for signal peptide cleavage at sites 1 and 2. No peptides corresponding to the signal peptide region (1–26) were identified. (D), Conclusion from mass spectrometry, showing the N-terminal sequence of GP5 with signal peptide (black), glycosylations (grey) and the positions of the neutralizing and the “decoy epitope”. Two GP5 species exist with signal peptide cleavage at sites 2 (top) and 1 (bottom), respectively.
Mentions: The observed migration pattern of deglycosylated GP5–HA is clear evidence for signal peptide cleavage; yet, the exact site of signal peptide cleavage cannot be derived from these data. We therefore employed mass spectrometry as a powerful tool to unequivocally identify proteins and protein fragments. Immunoprecipitation of cell-expressed GP5–HA does not yield enough material for mass spectrometry (data not shown). Therefore, we grew large quantities of PRRSV, strain VR-2332, in MARC-145 cells and partially purified the virus by PEG-8000 precipitation, ultracentrifugation and sucrose density gradient centrifugation (Fig. 7A). Virus proteins were subjected to PNGase F digestion to remove glycans and were separated by SDS-PAGE and stained with Coomassie. Deglycosylation is necessary to avoid heterogeneity in peptides due to the presence of differentially processed carbohydrate side chains. In addition, as a result of deglycosylation with PNGase F, asparagine is converted into aspartic acid and thus the occurrence of D instead of N is evidence that a certain glycosylation site is actually used. The band corresponding to GP5 (running at 16 kDa only after PNGase F digestion) was excised from the gel (see Fig. 7A). The band was verified to be GP5 by Western blot run in parallel, yielding a signal for GP5 at the same height as the excised band (Fig. 7A, right-hand panel). Two independent preparations were carried out.

Bottom Line: This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains.The results revealed that the signal peptide of GP5 is cleaved at two sites.This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Department of Veterinary Medicine, Free University Berlin, Berlin, Germany.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a "decoy epitope" located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the "decoy epitope" is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the "decoy epitope" sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".

Show MeSH
Related in: MedlinePlus