Pseudorabies virus (PrV) glycoproteins gll and gp5O are major constituents of the viral envelope and targets of neutralizing monoclonal antibodies. Both are homologs of essential glycoproteins found in herpes simplex virus, gB (gII) and gD (gp5O). We recently isolated a glI-negative PrV deletion mutant on complementing cell lines and established the essential character of gIl for PrV replication (I. Rauh, F.

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The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gIl 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 gIl 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 gIl gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gIl. We used a (T-galactosidase expression cassette inserted into a partially deleted cloned copy of the gIl gene for cotransfection with PrV DNA. gI-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 gll is indeed essential for PrV replication, since the gllmutants grew normally on gIl-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gIlmutants 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 gIl. gIl-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 gI-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 gll of PrV.

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