A glycoprotein gX-β-galactosidase fusion gene as insertional marker for rapid identification of pseudorabies virus mutants

Mettenleiter, Thomas C.; Rauh, Isabella

doi:10.1016/0166-0934(90)90043-f

Cited by 87 publications

(78 citation statements)

References 18 publications

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“…As can be seen in Fig. 2(a), compared to wt DNA mutant 1112 exhibits one additional fragment migrating between BamHI fragments 6 and 7 consisting of the fusion of fl-galactosidase sequences to fragment BamHI 10 which consequently does not appear (Mettenleiter et al, 1990). The luciferase recombinant A916 shows an additional fragment of approximately 9 kb migrating between BamHI fragments 4 and 5 (marked by arrow in Fig.…”

Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 81%

“…Purified DNA from the PrV gX-fl-galactosidaseexpressing mutant 1112 (Mettenleiter et al, 1990) was used for cotransfection with plasmid DNA using the calcium phosphate coprecipitation technique (Graham & van der Eb, 1973). After complete c.p.e, was observed, viral progeny was harvested and plaqueassayed under an agarose overlay containing 300 ~tg/ml of Bluo-gal (Gibco-BRL).…”

Section: Methodsmentioning

confidence: 99%

“…Viral progeny was screened for the appearance of white plaques under a Bluo-gal overlay. Plaques produced by the parental recombinant expressing flgalactosidase stained blue under these conditions (Mettenleiter & Rauh, 1990). Homologous recombination between TT-200 and the corresponding sequences in the genomic viral gX gene should result in loss of the flgalactosidase gene leading to a white plaque phenotype and acquisition of the luciferase gene.…”

Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 99%

“…To isolate a luciferase-expressing virus mutant DNA from PrV recombinant 1112 containing the fl-galactosidase gene under control of the gX promoter (Mettenleiter & Rauh, 1990) was used for cotransfection with plasmid TT-200. Viral progeny was screened for the appearance of white plaques under a Bluo-gal overlay.…”

Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 99%

“…Expression of fl-galactosidase from recombinant viral genomes can be analysed after addition of chromogenic substrates. This marker appears useful for selecting viral mutants (Mettenleiter et al, 1990) or identifying target cells in histological sections (Ho & Mocarski, 1988). However, sensitivity is limited and in infected organisms background problems often appear due to high endogenous fl-galactosidase activity (unpublished results).…”

Section: Introductionmentioning

confidence: 99%

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Firefly Luciferase as a Marker for Herpesvirus (pseudorabies virus) Replication in Vitro and in Vivo

Sz¹,

Mettenleiter

1991

Journal of General Virology

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Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 99%

Section: Isolation Of Luciferase-expressing Prvmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Firefly Luciferase as a Marker for Herpesvirus (pseudorabies virus) Replication in Vitro and in Vivo

Sz¹,

Mettenleiter

1991

Journal of General Virology

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Intracellular Processing of Pseudorabies Virus Glycoprotein M (gM): gM of Strain Bartha Lacks N-Glycosylation

1997

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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 of 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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Equine Herpesvirus 1 Mutants Devoid of Glycoprotein B or M Are Apathogenic for Mice but Induce Protection against Challenge Infection

Neubauer¹,

Beer

Brandmüller

et al. 1997

Virology

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Equine herpesvirus 1 (EHV-1) mutants devoid of the open reading frames (ORFs) of either glycoprotein (g) B or M were constructed and tested for their immunogenic potential in a murine model of EHV-1 infection. The mutant viruses were engineered using the virulent EHV-1 strain RacL11 or the modified live vaccine strain RacH by inserting the Escherichia coli LacZ gene into the viral ORFs. RacL11-infected mice showed signs typical of an EHV-1 infection, whereas mice infected with the EHV-1 gB- or gM-negative mutants or with RacH did not develop disease. No difference in the pathogenic potential of RacL11 gB- and gM-negative viruses was observed after application of either phenotypically completed or negative viruses. However, revertant RacL11 viruses in which the gB or gM gene had been restored caused EHV-1-related symptoms that were indistinguishable from those induced by RacL11. Mice that had been immunized with phenotypically negative gB- and gM-deficient EHV-1 were challenged with the RacL11 virus 25 days after immunization. Mock-immunized mice developed EHV-1 disease and high virus loads in their lungs were observed. In contrast, mice developed not exhibit EHV-1-caused disease. It was concluded (i) that deletion of either gB or gM abolished the virulence of strain RacL11 and (ii) that immunization with gB- or gM-negative EHV-1 elicited a protective immunity that was reflected by both virus-neutralizing antibodies and EHV-1-specific T-cells in spleens of immunized mice.

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A glycoprotein gX-β-galactosidase fusion gene as insertional marker for rapid identification of pseudorabies virus mutants

Cited by 87 publications

References 18 publications

Firefly Luciferase as a Marker for Herpesvirus (pseudorabies virus) Replication in Vitro and in Vivo

Firefly Luciferase as a Marker for Herpesvirus (pseudorabies virus) Replication in Vitro and in Vivo

Intracellular Processing of Pseudorabies Virus Glycoprotein M (gM): gM of Strain Bartha Lacks N-Glycosylation

Equine Herpesvirus 1 Mutants Devoid of Glycoprotein B or M Are Apathogenic for Mice but Induce Protection against Challenge Infection

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