Characterization of cell-binding properties of bovine herpesvirus 1 glycoproteins B, C, and D: identification of a dual cell-binding function of gB

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complexformation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from MATERIALS AND METHODSVirus and cell culture. The Colorado-1 strain of BHV-1 was obtained from the American Type Culture Collection. Virus was propagated in Madin-Darby bovine kidney (MDBK) cells as previously described (18).Construction of BHV-1 mutants K25 and K36. Details of the construction of the K25 and K36 BHV-1 mutants will be published later. A brief description follows. A cloned copy of the 8.4-kb HindIII-K fragment of BHV-1, which lies entirely within the short unique region of the genome, was subjected to partial digestion at random sites with HaeIII. Full-length linear molecules were isolated and ligated to a ␤-galactosidase expression unit. Recircularized plasmid molecules were transformed into Escherichia coli, and the positions of inserts within the K-fragment of individual clones were determined by restriction enzyme analysis. Clones containing inserts in the gI coding region (designated K36) and the gE coding region (designated K25) were cotransfected along with intact BHV-1 DNA into bovine cells, and viable recombinant viruses containing the inserts were recognized by their ability to form blue plaques.Production of gE-and gI-specific rabbit polyclonal antisera. gE and gI proteins were produced in E. coli as follows. DNA fragments from pBH144 (encoding the complete ORF of BHV-1 gI) and from pBH145B (encoding the complete ORF of ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins

Whitbeck

Knapp

Enquist

et al. 1996

“…Apoptosis is characterized morphologically by cell shrinkage, apoptotic-body formation, and condensation of the chromatin (6,20) and biochemically by fragmentation of DNA into oligonucleosomal DNA fragments (1,35). BHV-1 can induce apoptosis in peripheral blood mononuclear cells (PBMC) (11,16) and bovine B-lymphoma (BL-3) cells (10). The mechanism by which BHV-1 induces apoptotic cell death is not understood.…”

mentioning

confidence: 99%

Attachment but Not Penetration of Bovine Herpesvirus 1 Is Necessary To Induce Apoptosis in Target Cells

Hanon¹,

Meyer²,

Vanderplasschen³

et al. 1998

“…gB, gD, gH, and gL, which might act separately or, more likely, in combination, are essential for the entry process (17,47). Although this course of events appears to be generally accepted (21,38), it should be noted that the gB homologs of HSV-1 and BHV-1 also bind heparin (6,21,22), that the gD homolog of Marek's disease virus (MDV) is not essential for infectivity (39), and that the genome of varicellazoster virus (VZV) even lacks a gD homolog (48), indicating differences in the molecular mechanisms of the entry processes among alphaherpesviruses.…”

mentioning

confidence: 99%

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

Schröder

Linde

Fehler

et al. 1997

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.