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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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The majority of GP5–HA molecules in transfected cells is cleaved at site 2.MARC-145 cells were transfected with variants of GP5–HA or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (A) and after (B) PNGase F digestion to remove glycans. wt: GP5–HA wildtype; uncl.: Signal peptide (SP) cleavage completely blocked by mutation A26F, A29Y, A31F; cl.1: SP cleavage possible at site 1 (A26/V27) only (mutation A29S, A31Y); cl.2: SP cleavage at site 2 (A31/S32) only (mutation A26F, A29S). Black arrow: GP5–HA with cleaved SP, carrying three glycans, and/or GP5–HA comprising SP and two glycans; black arrow with plus sign: GP5–HA, SP cleaved, four glycans; white arrow: GP5–HA unprocessed/deglycosylated and containing the SP; grey and black arrowhead: GP5–HA with SP cleavage at site 1 or 2, respectively. (C), limited PNGase F digestion of GP5–HA uncl., performed and labeled as in Fig. 4. The protein carries two glycans.
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pone-0065548-g008: The majority of GP5–HA molecules in transfected cells is cleaved at site 2.MARC-145 cells were transfected with variants of GP5–HA or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (A) and after (B) PNGase F digestion to remove glycans. wt: GP5–HA wildtype; uncl.: Signal peptide (SP) cleavage completely blocked by mutation A26F, A29Y, A31F; cl.1: SP cleavage possible at site 1 (A26/V27) only (mutation A29S, A31Y); cl.2: SP cleavage at site 2 (A31/S32) only (mutation A26F, A29S). Black arrow: GP5–HA with cleaved SP, carrying three glycans, and/or GP5–HA comprising SP and two glycans; black arrow with plus sign: GP5–HA, SP cleaved, four glycans; white arrow: GP5–HA unprocessed/deglycosylated and containing the SP; grey and black arrowhead: GP5–HA with SP cleavage at site 1 or 2, respectively. (C), limited PNGase F digestion of GP5–HA uncl., performed and labeled as in Fig. 4. The protein carries two glycans.

Mentions: Wildtype GP5–HA and these mutants were expressed in MARC-145 cells and subjected to Western blot analysis before and after PNGase F digestion of lysates (Fig. 8). For GP5–HA uncl., a single band at the position of wildtype GP5–HA was detected before digestion with PNGase F (Fig. 8A, third lane), although a size increase by the retained signal peptide would be expected. However, after removal of glycans by PNGase F (Fig. 8B), this mutant exhibited a retarded SDS-PAGE mobility relative to GP5–HA wt and ran at the position expected for protein with uncleaved signal peptide (19 kDa). Limited PNGase F digestion revealed that GP5–HA uncl. carries only two glycans (Fig. 8C). It can be assumed that the glycosylation sites N30 and N33, albeit present in the protein, were not modified, most probably due to being held too close to the membrane by the uncleaved signal peptide that might function as a signal-anchor domain in that case.


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)

The majority of GP5–HA molecules in transfected cells is cleaved at site 2.MARC-145 cells were transfected with variants of GP5–HA or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (A) and after (B) PNGase F digestion to remove glycans. wt: GP5–HA wildtype; uncl.: Signal peptide (SP) cleavage completely blocked by mutation A26F, A29Y, A31F; cl.1: SP cleavage possible at site 1 (A26/V27) only (mutation A29S, A31Y); cl.2: SP cleavage at site 2 (A31/S32) only (mutation A26F, A29S). Black arrow: GP5–HA with cleaved SP, carrying three glycans, and/or GP5–HA comprising SP and two glycans; black arrow with plus sign: GP5–HA, SP cleaved, four glycans; white arrow: GP5–HA unprocessed/deglycosylated and containing the SP; grey and black arrowhead: GP5–HA with SP cleavage at site 1 or 2, respectively. (C), limited PNGase F digestion of GP5–HA uncl., performed and labeled as in Fig. 4. The protein carries two glycans.
© Copyright Policy
Related In: Results  -  Collection

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pone-0065548-g008: The majority of GP5–HA molecules in transfected cells is cleaved at site 2.MARC-145 cells were transfected with variants of GP5–HA or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (A) and after (B) PNGase F digestion to remove glycans. wt: GP5–HA wildtype; uncl.: Signal peptide (SP) cleavage completely blocked by mutation A26F, A29Y, A31F; cl.1: SP cleavage possible at site 1 (A26/V27) only (mutation A29S, A31Y); cl.2: SP cleavage at site 2 (A31/S32) only (mutation A26F, A29S). Black arrow: GP5–HA with cleaved SP, carrying three glycans, and/or GP5–HA comprising SP and two glycans; black arrow with plus sign: GP5–HA, SP cleaved, four glycans; white arrow: GP5–HA unprocessed/deglycosylated and containing the SP; grey and black arrowhead: GP5–HA with SP cleavage at site 1 or 2, respectively. (C), limited PNGase F digestion of GP5–HA uncl., performed and labeled as in Fig. 4. The protein carries two glycans.
Mentions: Wildtype GP5–HA and these mutants were expressed in MARC-145 cells and subjected to Western blot analysis before and after PNGase F digestion of lysates (Fig. 8). For GP5–HA uncl., a single band at the position of wildtype GP5–HA was detected before digestion with PNGase F (Fig. 8A, third lane), although a size increase by the retained signal peptide would be expected. However, after removal of glycans by PNGase F (Fig. 8B), this mutant exhibited a retarded SDS-PAGE mobility relative to GP5–HA wt and ran at the position expected for protein with uncleaved signal peptide (19 kDa). Limited PNGase F digestion revealed that GP5–HA uncl. carries only two glycans (Fig. 8C). It can be assumed that the glycosylation sites N30 and N33, albeit present in the protein, were not modified, most probably due to being held too close to the membrane by the uncleaved signal peptide that might function as a signal-anchor domain in that case.

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