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

Abstract

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 ...

The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

“…Previous studies involving nonpolarized cell lines such as L cells, Vero cells, and human R970 and HeLa cells indicated that gE moved to the cell surface without gI (5,19,45). Here, we found that gE accumulated in perinuclear, cytoplasmic vesicles in epithelial cells, consistent with reports that HSV gE and alphaherpesvirus gE homologues require coexpression of gI for efficient transport to the cell surface in some but not all types of cells (29,33,49,50,53). Therefore, there appears to be retention in the endoplasmic reticulum and possibly misfolding of gE when the glycoprotein is expressed without gI, at least in some types of cells.…”

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1

“…When compared with the internal control PRV gC protein, the total amount of BHV-1 gI produced from PRV-AK7 was 15-fold higher than that produced from PRV-AK1, as determined by densitometry (data not shown). Despite the overproduction of gI by PRV-AK7, the kinetics of synthesis and processing were not significantly different than those found for PRV-AK1 or for wild-type BHV-1 in MDBK cells (37).…”

“…PRV gC and gB were used as internal controls to measure relative time of expression and quantity for comparison between samples. In a previous study, we determined that the BHV-1 gE 92-kDa protein was the mature species, while the 84-kDa species was the precursor species (37). Likewise for BHV-1 gI, the 62-kDa species is the mature form, and the 40-kDa species is the precursor of BHV-1 gI.…”

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 schematic representation of the prototype orientation of the BHV-1 genome is shown above the HindIII restriction fragment map of BHV-1 strain Schönböken (7,27). The wild-type HindIII L fragment is enlarged, and the location and direction of transcription of genes encoding the putative protein kinase (PK) and glycoproteins G (gG), D (gD), I (gI), and E (gE) are indicated by arrows (15,25,40,45). Relevant restriction enzyme cleavage sites are marked.…”

“…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).…”