From essential to beneficial: glycoprotein D loses importance for replication of bovine herpesvirus 1 in cell culture

Abstract

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1) has been shown to be an essential component of virions involved in virus entry. gD expression in infected cells is also required for direct cell-to-cell spread. Therefore, BHV-1 gD functions are identical in these aspects to those of herpes simplex virus 1 (HSV-1) gD. In contrast, the gD homolog of pseudorabies virus (PrV), although essential for penetration, is not necessary for direct cell-to-cell spread. Cocultivation of cells infected with phenotypically gD-complemented gD ؊ mutant BHV-1/ 80-221 with noncomplementing cells resulted in the isolation of the cell-to-cell-spreading gD-negative mutant ctcs ؉ BHV-1/80-221, which was present in the gD-null BHV-1 stocks. ctcs ؉ BHV-1/80-221 could be propagated only by mixing infected with uninfected cells, and virions released into the culture medium were noninfectious. Marker rescue experiments revealed that a single point mutation in the first position of codon 450 of the glycoprotein H open reading frame, resulting in a glycine-to-tryptophan exchange, enabled complementation of the gD function for cell-to-cell spread. After about 40 continuous passages of ctcs ؉ BHV-1/80-221-infected cells with noninfected cells, the plaque morphology in the cultures started to change from roundish to comet shaped. Cells from such plaques produced infectious gD ؊ virus, named gD ؊ infBHV-1, which entered cells much more slowly than wild-type BHV-1. In contrast, integration of the gD gene into the genomes of gD ؊ infBHV-1 and ctcs ؉ BHV-1/80-221 resulted in recombinants with accelerated penetration in comparison to wild-type virions. In summary, our results demonstrate that under selective conditions, the function of BHV-1 gD for direct cell-to-cell spread and entry into cells can be compensated for by mutations in other viral (glyco)proteins, leading to the hypothesis that gD is involved in formation of penetration-mediating complexes in the viral envelope of which gH is a component. Together with results for PrV, varicella-zoster virus, which lacks a gD homolog, and Marek's disease virus, whose gD homolog is not essential for infectivity, our data may open new insights into the evolution of alphaherpesviruses.

Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

“…A similar mutant could also be obtained for BHV-1 (40). For BHV-1, it has been shown that mutations in at least gB and gH contribute to gD-independent infectivity (40,41). The data presented here for PrV correlate with these earlier findings.…”

“…70-fold) reduction in specific infectivity in cell culture (17). A similar mutant has also been isolated from BHV-1 (40). This phenotype is at least partly due to compensatory mutations in gB and gH (references 40 and 41 and this paper).…”

“…Recently, we isolated an infectious gD-negative PrV mutant, PrV-gD Ϫ Pass (39). A similar mutant could also be obtained for BHV-1 (40). For BHV-1, it has been shown that mutations in at least gB and gH contribute to gD-independent infectivity (40,41).…”

The Class II Membrane Glycoprotein G of Bovine Respiratory Syncytial Virus, Expressed from a Synthetic Open Reading Frame, Is Incorporated into Virions of Recombinant Bovine Herpesvirus 1

“…The cells were grown in Dulbecco's minimum essential medium supplemented with 5% fetal calf serum (FCS), 100 U of penicillin per ml, 100 g of streptomycin per ml, and 0.35 mg of L-glutamine per ml. The gD-negative mutant BHV-1/80-221 was propagated on the constitutively gD-expressing cell line BU-Dorf as described previously (38).…”

“…As with other large DNA viruses, interest exists in the use of recombinant BHV-1 as an improved live vaccine against BHV-1 infection (1,21,42) or as a vector for bi-or multivalent vaccines against BHV-1 and additional bovine pathogens (17,18). To date, incorporation of heterologous genes into the genome of BHV-1 has concentrated mainly on the expression of the procaryotic lacZ gene to identify essential and nonessential genes or as a reporter gene for analytical studies (3,8,12,15,20,29,37,38,45). Recently, BHV-1 has been used to express biologically active bovine interleukins (21,32) and glycoproteins of pseudorabiesvirus (19,31).…”