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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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Conclusion – Major fraction of GP5 from PRRSV type 2 does not contain the “decoy epitope”.Schematic representation of GP5 fractions as evidenced in this study. The signal peptide of GP5 is predominantly cleaved at site 2 (A26/V27; black arrow), but is also cleaved in minor quantities at site 1 (A26/V27, thin grey arrow). The “decoy epitope” (magenta) is preserved only in GP5 cleaved at site 1. The neutralizing epitope (green) is present in either case. Heterogeneity occurs also at non-conserved glycosylation sites (red). The fraction of GP5 with the “decoy epitope” contains carbohydrates at either both N30 and N33, or only at N33 or none of these sites. A subfraction of site 2-cleaved GP5 does not contain a carbohydrate at N33. Conserved glycosylation sites are in brown.
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pone-0065548-g009: Conclusion – Major fraction of GP5 from PRRSV type 2 does not contain the “decoy epitope”.Schematic representation of GP5 fractions as evidenced in this study. The signal peptide of GP5 is predominantly cleaved at site 2 (A26/V27; black arrow), but is also cleaved in minor quantities at site 1 (A26/V27, thin grey arrow). The “decoy epitope” (magenta) is preserved only in GP5 cleaved at site 1. The neutralizing epitope (green) is present in either case. Heterogeneity occurs also at non-conserved glycosylation sites (red). The fraction of GP5 with the “decoy epitope” contains carbohydrates at either both N30 and N33, or only at N33 or none of these sites. A subfraction of site 2-cleaved GP5 does not contain a carbohydrate at N33. Conserved glycosylation sites are in brown.

Mentions: Here we show that the signal peptide is cleaved from GP5 of the American PRRSV type 2 reference strain VR-2332, both upon in vitro translation in the presence of microsomes (Fig. 2) as well as in transfected cells (Fig. 3). Processing was not affected by deletion and insertion of glycosylation sites in the vicinity of the cleavage site, which also occurs in natural virus strains (Fig. 2+3). Likewise, the signal peptide was also cleaved from different GP5 variants derived from natural virus strains (attenuated and virulent ones). These differ regarding the amino acids in the vicinity of the signal peptide cleavage site and the number of glycans (Fig. 6). In addition, a variety of porcine cell lines processed GP5 in the same manner, as far as discernible by SDS-PAGE (Fig. 5). Mass spectrometry of peptides derived from the GP5 of virus particles and from a recombinant GP5/M dimer expressed in insect cells unequivocally demonstrated that the signal peptide is cleaved from every GP5 molecule (Fig. 7). Intriguingly, two different cleavage sites were identified, which are identical to the sites predicted by SignalP 4.0. Thus, there are probably two fractions of GP5 molecules present in virus particles, one with and one without the “decoy epitope”. By comparing the wildtype GP5 probe to mutants where either signal peptide cleavage site 1 or 2 was blocked, we obtained circumstantial evidence that the largest part of wildtype GP5 is cleaved at site 2 and will thus not contain the “decoy epitope” (Fig. 8). This is schematically displayed in Fig. 9.


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)

Conclusion – Major fraction of GP5 from PRRSV type 2 does not contain the “decoy epitope”.Schematic representation of GP5 fractions as evidenced in this study. The signal peptide of GP5 is predominantly cleaved at site 2 (A26/V27; black arrow), but is also cleaved in minor quantities at site 1 (A26/V27, thin grey arrow). The “decoy epitope” (magenta) is preserved only in GP5 cleaved at site 1. The neutralizing epitope (green) is present in either case. Heterogeneity occurs also at non-conserved glycosylation sites (red). The fraction of GP5 with the “decoy epitope” contains carbohydrates at either both N30 and N33, or only at N33 or none of these sites. A subfraction of site 2-cleaved GP5 does not contain a carbohydrate at N33. Conserved glycosylation sites are in brown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675037&req=5

pone-0065548-g009: Conclusion – Major fraction of GP5 from PRRSV type 2 does not contain the “decoy epitope”.Schematic representation of GP5 fractions as evidenced in this study. The signal peptide of GP5 is predominantly cleaved at site 2 (A26/V27; black arrow), but is also cleaved in minor quantities at site 1 (A26/V27, thin grey arrow). The “decoy epitope” (magenta) is preserved only in GP5 cleaved at site 1. The neutralizing epitope (green) is present in either case. Heterogeneity occurs also at non-conserved glycosylation sites (red). The fraction of GP5 with the “decoy epitope” contains carbohydrates at either both N30 and N33, or only at N33 or none of these sites. A subfraction of site 2-cleaved GP5 does not contain a carbohydrate at N33. Conserved glycosylation sites are in brown.
Mentions: Here we show that the signal peptide is cleaved from GP5 of the American PRRSV type 2 reference strain VR-2332, both upon in vitro translation in the presence of microsomes (Fig. 2) as well as in transfected cells (Fig. 3). Processing was not affected by deletion and insertion of glycosylation sites in the vicinity of the cleavage site, which also occurs in natural virus strains (Fig. 2+3). Likewise, the signal peptide was also cleaved from different GP5 variants derived from natural virus strains (attenuated and virulent ones). These differ regarding the amino acids in the vicinity of the signal peptide cleavage site and the number of glycans (Fig. 6). In addition, a variety of porcine cell lines processed GP5 in the same manner, as far as discernible by SDS-PAGE (Fig. 5). Mass spectrometry of peptides derived from the GP5 of virus particles and from a recombinant GP5/M dimer expressed in insect cells unequivocally demonstrated that the signal peptide is cleaved from every GP5 molecule (Fig. 7). Intriguingly, two different cleavage sites were identified, which are identical to the sites predicted by SignalP 4.0. Thus, there are probably two fractions of GP5 molecules present in virus particles, one with and one without the “decoy epitope”. By comparing the wildtype GP5 probe to mutants where either signal peptide cleavage site 1 or 2 was blocked, we obtained circumstantial evidence that the largest part of wildtype GP5 is cleaved at site 2 and will thus not contain the “decoy epitope” (Fig. 8). This is schematically displayed in Fig. 9.

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