Functional Analysis of the Transmembrane Anchor Region of Bovine Herpesvirus 1 Glycoprotein gB

Abstract

In herpesviruses, homologues of glycoprotein B (gB) are essential membrane proteins which are involved in fusion. However, there is no clear evidence regarding the location of the fusogenic domain on gB. By using bovine herpesvirus 1 (BHV-1) as a model, we studied the relationship between the structure and the fusogenic activity of gB. This was achieved by expressing genes of different gB derivatives containing specific truncations at the end of segments 2 or 3 of the transmembrane region in Madin-Darby bovine kidney cells under the control of the bovine heat-shock protein hsp70A gene promoter. All expressed gB products were structurally similar to authentic gB. One truncated form of gB, gBt, which contains residues 1-763, was efficiently secreted. However, gBtM (residues 1-807), which includes the first two segments at the carboxyl terminus, showed unstable retention on the cell surface, whereas gBtMA (residues 1 829), which contains all three membrane-spanning segments, was mostly intracellularly retained with some unstable surface anchorage. Another truncated gB, gBtDAF, which has gB residues 1-763 (gBt) and a human decay-accelerating factor (DAF) carboxyl tail, was also expressed. The DAF fragment provided a signal for the addition of a glycosyl phosphatidylinositol-based membrane anchor, which could target the gBt chimeric protein on the cell membrane. Immunofluorescence staining and pulse-chase kinetic studies support the theory that gBtM, gBtMA, and gBtDAF are retained on nuclear and cellular membranes via different segments of the transmembrane region or the DAF fragment, respectively. For the cells expressing gBt or gBtM, no cell fusion was observed, whereas cells expressing gBtMA clearly showed fusion. However, in gBtDAF cells, the overexpression and cellular accumulation of recombinant gB products did not cause fusion either, which supports our contention that the fusion phenomenon in gBtMA cells is caused by the fusogenic activity of the expressed gBtMA. With the help of sequence analysis, our results indicate that segment 2 of the transmembrane anchor region might be a fusogenic domain, whereas the real anchor is segment 3.

The Immunogenicity and Protective Efficacy of Bovine Herpesvirus 1 Glycoprotein D plus Emulsigen Are Increased by Formulation with CpG Oligodeoxynucleotides

“…It was expressed in MDBK cells under regulation of the bovine heat shock 70A (hsp70) gene promoter (21) and purified as previously discussed (48). Authentic gD (45) and tgB (24) were produced, processed, and purified as described elsewhere.…”

Gene Gun-Mediated DNA Immunization Primes Development of Mucosal Immunity against Bovine Herpesvirus 1 in Cattle

“…Plasmids pSLIAgB and pSLIAtgB were constructed by cloning the genes coding for gB and a truncated version of gB (tgB) into pSL301 (Invitrogen, Carlsbad, Calif.) as described previously (4). tgB lacks the transmembrane anchor and cytoplasmic domain and is therefore secreted from transfected cells (29). A plasmid encoding green fluorescent protein (GFP) was obtained from Quantum Technologies, Laval, Quebec, Canada, to be used for detection of protein expression.…”

Duck Enteritis Virus Glycoprotein D and B DNA Vaccines Induce Immune Responses and Immunoprotection in Pekin Ducks

“…Glycoprotein B (gB) is one of the major glycoproteins in the viral envelope and plasma membrane of virus-infected cells. The gB is essential for attachment and penetration of free virions [13], involving fusion between the virion envelope and the cellular plasma membrane [14], as well as direct transmission of infectivity from primary infected cells to neighboring non-infected cells [15], [16]. In many herpesviruses, the gB homolog has been reported to elicit neutralizing antibodies in mammals and birds [17], [18], [19], [20], [21], [22], [23], [24].…”