Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.
The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is integrated into the PrV envelope and can functionally replace glycoprotein gII of PrV.
Rauh
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I
Federal Research Centre for Virus Diseases of Animals, Tübingen, Federal Republic of Germany.
Weiland
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Fehler
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Keil
G M
GM
Mettenleiter
T C
TC
D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D005814:Genes, Viral; D005816:Genetic Complementation Test; D007242:Herpesvirus 1, Bovine / Q000235:genetics*; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000648:ultrastructure; D000090023:Metallothionein 3; D016253:Microscopy, Immunoelectron; D016296:Mutagenesis; D010957:Plasmids; D015183:Restriction Mapping; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014771:Virion / Q000235:genetics / Q000648:ultrastructure
D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; C042966:pseudorabies virus glycoproteins