Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gI, gIII, and gIV

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%

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

Whitbeck

Knapp

Enquist

et al. 1996

“…Purification of virions. BHV-1 virions were purified from the supernatant of virus-infected MDBK cell culture by an Na-K tartrate gradient, as described before (16). Briefly, cell supernatant containing virus was clarified by low-speed centrifugation at 1,000 ϫ g. Virus was harvested by pelleting through 30% sucrose in phosphate-buffered saline (PBS) at 100,000 ϫ g for 60 min.…”

Section: Methodsmentioning

confidence: 99%

“…Subconfluent MDBK cells grown in T-25 flasks were infected with virus at a multiplicity of infection of 5. Radiolabelling with [ 3 H]glucosamine (100 Ci/ml in glucose-deficient minimum essential medium supplemented with 10% dialyzed FBS; Amersham), [ 3 H]myristic acid (25 Ci/ml in minimum essential medium supplemented with 10% dialyzed FBS; Amersham), or Tran 35 S-label (50 Ci/ml in a methionine-cysteine-deficient minimum essential medium supplemented with 10% FBS; ICN) was carried out for 16 h beginning 1 h postinfection; radiolabelling with [ 32 P]phosphoric acid (100 Ci/ml in a phosphate-deficient medium supplemented with 10% dialyzed FBS; ICN) was carried out at 16 h postinfection for 1 h. Immunoprecipitation, following labelling, was carried out as previously reported (16) with modifications. The radiolabelled cells from each T-25 flask were lysed in 300 l of RIPA buffer composed of 0.15 M NaCl, 10 mM Tris-HCl, 1% deoxycholic acid, 1% Nonidet P-40 (NP-40), 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, and 0.5 mg of aprotinin per ml (pH 8.0) at 4ЊC for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein

Liang

Chow

Raggo

et al. 1996

Self Cite

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-1 UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms: monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [ 3 H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.

“…A series of recent reports dealing with the entry of herpes simplex virus type 1 (HSV-1), pseudorabies virus (PrV), and bovine herpesvirus 1 (BHV-1) showed that infection of target cells is a complicated process involving a cascade of events. Following initial interaction between glycoprotein C (gC) and heparan sulfate on the cell surface (8,12,13,23,24,30,31,35,51), gD presumably interacts with cellular receptors, resulting in stable attachment (15,46). Subsequent pH-independent fusion between the target cell membrane and the viral envelope results in release of the viral nucleocapsid into the cytoplasm of the host cell (50).…”

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.