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. 1998 May;72(5):3804-11.
doi: 10.1128/JVI.72.5.3804-3811.1998.

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

G Kühnle  1 , A Heinze, J Schmitt, K Giesow, G Taylor, I Morrison, F A Rijsewijk, J T van Oirschot, G M Keil
Affiliations

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

G Kühnle et al. J Virol. 1998 May.

Abstract

The bovine herpesvirus 1 (BHV-1) recombinants BHV-1/eG(ori) and BHV-1/eG(syn) were isolated after insertion of expression cassettes which contained either a genomic RNA-derived cDNA fragment (BHV-1/eG(ori)) or a modified, chemically synthesized open reading frame (ORF) (BHV-1/eG(syn)), which both encode the attachment glycoprotein G of bovine respiratory syncytial virus (BRSV), a class II membrane glycoprotein. Northern blot analyses and nuclear runoff transcription experiments indicated that transcripts encompassing the authentic BRSV G ORF were unstable in the nucleus of BHV-1/eG(ori)-infected cells. In contrast, high levels of BRSV G RNA were detected in BHV-1/eG(syn)-infected cells. Immunoblots showed that the BHV-1/eG(syn)-expressed BRSV G glycoprotein contains N- and O-linked carbohydrates and that it is incorporated into the membrane of infected cells and into the envelope of BHV-1/eG(syn) virions. The latter was also demonstrated by neutralization of BHV-1/eG(syn) infectivity by monoclonal antibodies or polyclonal anti-BRSV G antisera and complement. Our results show that expression of the BRSV G glycoprotein by BHV-1 was dependent on the modification of the BRSV G ORF and indicate that incorporation of class II membrane glycoproteins into BHV-1 virions does not necessarily require BHV-1-specific signals. This raises the possibility of targeting heterologous polypeptides to the viral envelope, which might enable the construction of BHV-1 recombinants with new biological properties and the development of improved BHV-1-based live and inactivated vector vaccines.

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Figures

FIG. 1
FIG. 1
Construction of BHV-1 recombinants. 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. The respective HindIII fragment of the gD− lacZ+ mutant BHV-1/80–221 is depicted below. The location and direction of transcription of the lacZ cassette (dotted area, not drawn to scale) that replaces the gD ORF is indicated by an arrow. Underneath, a diagram of the integration fragment contained in the recombination vectors is shown. The gD TATA and gD poly(A) segments indicate BHV-1 sequences representing the gD promoter and the gD polyadenylation signal, respectively, which provide homologous regions for recombination. In this study, transcription of the BRSV Gori or Gsyn ORF (BRSVG ORF) was directed by the MCMV ie1 enhancer-promoter element (6) in transient-expression experiments or by the MCMV e1 promoter (2) in recombinant BHV-1 infected cells. Transcription of the BHV-1 gD ORF which is followed by the MCMV ie2 poly(A) signal (28) is under the control of the authentic gD promoter.
FIG. 2
FIG. 2
Cell-free translation of cRNAs encompassing the BRSV Gori or Gsyn ORF. 35S-labeled proteins translated in vitro from RNA transcribed in vitro from plasmids pSPGori (lane 2) or pSPGsyn (lane 3) were size separated in SDS–10% polyacrylamide gels and visualized by fluorography. In lane 1, proteins synthesized from RNA transcribed from pSPGori antisense to the BRSV G glycoprotein ORF were separated. The apparent molecular mass of the translation products, indicated in kilodaltons on the left, was calculated from the migration of 14C-methylated protein molecular mass standards run on the same gel.
FIG. 3
FIG. 3
Comparison of the nucleotide sequences of the BRSV G glycoprotein cDNA ORF (upper sequence) and the Gsyn ORF (lower sequence). Exchanged nucleotides are in boldface type, and the deduced BRSV G glycoprotein amino acid sequence is shown in the single-letter code.
FIG. 4
FIG. 4
Identification of transcripts encompassing the Gsyn ORF. Whole-cell RNA from cells infected with BHV-1/eGsyn (lanes 1, 3, 5, and 7) and BHV-1/eGori (lanes 2, 4, 6, and 8) was prepared at 6 h p.i., and 5-μg samples were transferred to nitrocellulose after 1% agarose gel electrophoresis. The filters were hybridized to 32P-labeled DNA from the BRSV-Gsyn ORF (lanes 1 and 5), the BRSV-Gori ORF (lanes 2 and 6), and the BHV-1 gD ORF (lanes 3, 4, 7, and 8). Bound radioactivity was visualized by autoradiography. Lanes 5 to 8 are longer exposures of lanes 1 to 4. Transcript sizes are indicated in kilobases.
FIG. 5
FIG. 5
BHV-1-encoded BRSV G glycoprotein is expressed on the cell surface. MDBK cell cultures were infected with approximately 100 PFU of BHV-1/Schö, BHV-1/eGsyn, and BHV-1/eGori, fixed after the development of plaques, and used for indirect immunofluorescence with BHV-1 gD-specific MAb 21/3/3, BRSV G glycoprotein-specific MAb 20, and the anti-VacGsyn serum. Bound antibodies were visualized by staining with DTAF-conjugated goat anti-species immunoglobulin G.
FIG. 6
FIG. 6
Identification of the BHV-1/eGsyn-expressed BRSV G glycoprotein. MDBK cells were infected with BHV-1/Schö (lanes 1 and 5), BHV-1/eGori (lanes 2 and 6), and BHV-1/eGsyn (lanes 3, 4, 7, and 8). Proteins from infected cells, harvested at 10 h p.i. (lanes 1 to 4), from purified virions (lanes 5 to 7), and from cell culture medium (lane 8), were analyzed by immunoblotting with BRSV G glycoprotein-specific MAb 20. Proteins shown in lane 4 were from cells incubated with cycloheximide (100 μg/ml) for 2 h before lysis. Apparent molecular masses are indicated in kilodaltons.
FIG. 7
FIG. 7
Evidence that BHV-1-expressed G glycoprotein contains N- and O-linked sugars. (a and b) Purified BHV-1/eGsyn virions were resuspended in PBS plus 0.5% NP-40 and digested overnight at 37°C with neuraminidase (lanes 2), neuraminidase and O-glycosidase (lanes 3), N-glycosidase F (lanes 4), and without enzyme (lanes 1). Digestion products were analyzed by immunoblotting with G glycoprotein-specific MAb 20 (a) or a polyclonal, BHV-1 gD-specific rabbit antiserum (b). (c) Purified, [35S]methionine-labeled BHV-1/Schö virions were lysed and incubated with gD-specific MAb 21/3/3. Immunoprecipitated proteins were incubated as described above, separated on SDS–10% polyacrylamide gels, and visualized by fluorography. The apparent molecular masses, indicated in kilodaltons on the left, were calculated from the migration of prestained (a and b) or 14C-methylated protein molecular mass standards (c) run on the respective gel.
FIG. 8
FIG. 8
BHV-1/eGsyn virions are susceptible to neutralization by antibodies against the BRSV G glycoprotein. Approximately 200 PFU of BHV-1/eGori was incubated with 20% fetal calf serum plus complement or serial dilutions of anti-VacGsyn with complement (open squares) or the anti-BRSV hyperimmune serum with complement (open circles), and about 160 PFU of BHV-1/eGsyn was incubated with 20% FCS and complement or with serial dilutions of anti-VacGsyn and complement (solid squares), MAb 57 with complement (stars), anti-BRSV serum 2106 without complement (solid triangles, no complement added to the FCS control), or anti-BRSV serum 2106 with complement (solid circles). After 60 min at 37°C, the virions were plated on MDBK cells and the cultures were overlaid with semisolid medium. Plaques were counted 3 days later. The plaque count of each FCS control was defined as 100% neutralization-resistant infectivity. Serum dilutions are indicated.
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