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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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Processing analysis of cell expressed GP5–HA to reveal glycan-independent signal peptide cleavage.CHO-K1 (A, B) and MARC-145 (C, D; permissive for PRRSV) were transfected with plasmids encoding GP5–HA as wildtype (wt) and mutants with deleted or added glycosylation sites near the signal peptide cleavage site; Ø, empty plasmid control. Cell lysates were subjected to SDS-PAGE and Western blot (anti-HA tag) before (A, C) and after (B, D) deglycosylation with PNGase F. Molecular weight marker given on the left-hand side; arrows indicate sizes of unprocessed GP5–HA (white), glycosylated protein (black: wildtype glycosylation; grey: lacking one glycan; black/+: with one additional glycan), and deglycosylated protein without signal peptide (black arrowhead). In B and D, in vitro-generated GP5–HA (in the absence of microsomes, thus intrinsically unprocessed, i.e. not glycosylated and still containing the signal peptide, cf. Fig. 2) is shown in the rightmost lane for size comparison. Deglycosylated GP5–HA and all variants mostly ran faster than unprocessed protein.
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pone-0065548-g003: Processing analysis of cell expressed GP5–HA to reveal glycan-independent signal peptide cleavage.CHO-K1 (A, B) and MARC-145 (C, D; permissive for PRRSV) were transfected with plasmids encoding GP5–HA as wildtype (wt) and mutants with deleted or added glycosylation sites near the signal peptide cleavage site; Ø, empty plasmid control. Cell lysates were subjected to SDS-PAGE and Western blot (anti-HA tag) before (A, C) and after (B, D) deglycosylation with PNGase F. Molecular weight marker given on the left-hand side; arrows indicate sizes of unprocessed GP5–HA (white), glycosylated protein (black: wildtype glycosylation; grey: lacking one glycan; black/+: with one additional glycan), and deglycosylated protein without signal peptide (black arrowhead). In B and D, in vitro-generated GP5–HA (in the absence of microsomes, thus intrinsically unprocessed, i.e. not glycosylated and still containing the signal peptide, cf. Fig. 2) is shown in the rightmost lane for size comparison. Deglycosylated GP5–HA and all variants mostly ran faster than unprocessed protein.

Mentions: We transfected CHO-K1 cells, which are known for good transfection efficiencies and expression rates, as well as MARC-145 cells, which are permissive for PRRSV and therefore particularly relevant for the assessment of GP5 processing [43]. SDS-PAGE and Western blot of cell lysates after transfection showed that all GP5–HA variants were expressed and apparently glycosylated (Fig. 3A, C). The comparison of electrophoretic mobilities between mutants shows that GP5–HA N30S ran at the same height as the corresponding wildtype. The size of the N33S as well as the N30S, N33S mutant appears to be reduced by roughly 2.5 kDa (one glycan). Limited digestion of GP5–HA wt with PNGase F digestion showed that GP5 comprised three glycans (Fig. 4). Thus, all the potential glycosylation sites except N30 (i.e., N33, N44, and N51) were indeed used, which is in line with previous investigations on PRRSV-GP5 [44]. The major band of GP5–HA D34N is increased in size by one additional glycan, showing that the additionally introduced glycosylation site is used. The (weaker) band at the height of wildtype protein indicates that this additional glycosylation is not realized in every molecule, probably because the glycosylation sequons of N33 (N33N34S35) and N34 (N34S35S36) overlap.


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)

Processing analysis of cell expressed GP5–HA to reveal glycan-independent signal peptide cleavage.CHO-K1 (A, B) and MARC-145 (C, D; permissive for PRRSV) were transfected with plasmids encoding GP5–HA as wildtype (wt) and mutants with deleted or added glycosylation sites near the signal peptide cleavage site; Ø, empty plasmid control. Cell lysates were subjected to SDS-PAGE and Western blot (anti-HA tag) before (A, C) and after (B, D) deglycosylation with PNGase F. Molecular weight marker given on the left-hand side; arrows indicate sizes of unprocessed GP5–HA (white), glycosylated protein (black: wildtype glycosylation; grey: lacking one glycan; black/+: with one additional glycan), and deglycosylated protein without signal peptide (black arrowhead). In B and D, in vitro-generated GP5–HA (in the absence of microsomes, thus intrinsically unprocessed, i.e. not glycosylated and still containing the signal peptide, cf. Fig. 2) is shown in the rightmost lane for size comparison. Deglycosylated GP5–HA and all variants mostly ran faster than unprocessed protein.
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Related In: Results  -  Collection

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pone-0065548-g003: Processing analysis of cell expressed GP5–HA to reveal glycan-independent signal peptide cleavage.CHO-K1 (A, B) and MARC-145 (C, D; permissive for PRRSV) were transfected with plasmids encoding GP5–HA as wildtype (wt) and mutants with deleted or added glycosylation sites near the signal peptide cleavage site; Ø, empty plasmid control. Cell lysates were subjected to SDS-PAGE and Western blot (anti-HA tag) before (A, C) and after (B, D) deglycosylation with PNGase F. Molecular weight marker given on the left-hand side; arrows indicate sizes of unprocessed GP5–HA (white), glycosylated protein (black: wildtype glycosylation; grey: lacking one glycan; black/+: with one additional glycan), and deglycosylated protein without signal peptide (black arrowhead). In B and D, in vitro-generated GP5–HA (in the absence of microsomes, thus intrinsically unprocessed, i.e. not glycosylated and still containing the signal peptide, cf. Fig. 2) is shown in the rightmost lane for size comparison. Deglycosylated GP5–HA and all variants mostly ran faster than unprocessed protein.
Mentions: We transfected CHO-K1 cells, which are known for good transfection efficiencies and expression rates, as well as MARC-145 cells, which are permissive for PRRSV and therefore particularly relevant for the assessment of GP5 processing [43]. SDS-PAGE and Western blot of cell lysates after transfection showed that all GP5–HA variants were expressed and apparently glycosylated (Fig. 3A, C). The comparison of electrophoretic mobilities between mutants shows that GP5–HA N30S ran at the same height as the corresponding wildtype. The size of the N33S as well as the N30S, N33S mutant appears to be reduced by roughly 2.5 kDa (one glycan). Limited digestion of GP5–HA wt with PNGase F digestion showed that GP5 comprised three glycans (Fig. 4). Thus, all the potential glycosylation sites except N30 (i.e., N33, N44, and N51) were indeed used, which is in line with previous investigations on PRRSV-GP5 [44]. The major band of GP5–HA D34N is increased in size by one additional glycan, showing that the additionally introduced glycosylation site is used. The (weaker) band at the height of wildtype protein indicates that this additional glycosylation is not realized in every molecule, probably because the glycosylation sequons of N33 (N33N34S35) and N34 (N34S35S36) overlap.

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