Intracellular processing of pseudorabies virus glycoprotein M (IgM): gM of strain Bartha lacks N-glycosylation

J・m Dijkstra, Thomas C. Mettenleiter, Barbara G. Klupp

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Genes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM-PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role, To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation or several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation, Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in Virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture.

Original languageEnglish
Pages (from-to)113-122
Number of pages10
JournalVirology
Volume237
Issue number1
DOIs
Publication statusPublished - 13-10-1997

Fingerprint

Suid Herpesvirus 1
Glycosylation
Immunoglobulin M
Consensus Sequence
Polysaccharides
Herpesviridae
Proteins
Cell Culture Techniques
pseudorabies virus glycoproteins
Genes
Viruses
Glycoside Hydrolases
Human Herpesvirus 1
Virus Diseases
Virion
Sequence Analysis
Digestion
Glycoproteins
Vaccines
Western Blotting

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Dijkstra, J・m ; Mettenleiter, Thomas C. ; Klupp, Barbara G. / Intracellular processing of pseudorabies virus glycoprotein M (IgM) : gM of strain Bartha lacks N-glycosylation. In: Virology. 1997 ; Vol. 237, No. 1. pp. 113-122.
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abstract = "Genes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM-PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role, To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation or several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation, Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in Virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture.",
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Intracellular processing of pseudorabies virus glycoprotein M (IgM) : gM of strain Bartha lacks N-glycosylation. / Dijkstra, J・m; Mettenleiter, Thomas C.; Klupp, Barbara G.

In: Virology, Vol. 237, No. 1, 13.10.1997, p. 113-122.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Intracellular processing of pseudorabies virus glycoprotein M (IgM)

T2 - gM of strain Bartha lacks N-glycosylation

AU - Dijkstra, J・m

AU - Mettenleiter, Thomas C.

AU - Klupp, Barbara G.

PY - 1997/10/13

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N2 - Genes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM-PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role, To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation or several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation, Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in Virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture.

AB - Genes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM-PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role, To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation or several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation, Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in Virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture.

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