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

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 notnecessarily 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.

A Hepadnavirus Regulatory Element Enhances Expression of a Type 2 Bovine Viral Diarrhea Virus E2 Protein from a Bovine Herpesvirus 1 Vector

“…Although the E2 protein was expressed to a significant extent in BHV1, even in the absence of the WPRE, it is likely that a BHV1 recombinant containing the WPRE would elicit a higher E2 antibody response in a vaccinated animal than one lacking the WPRE. However, the use of the WPRE in BHV1 recombinants assumes its greatest importance with respect to foreign genes that are normally expressed at undetectable levels in BHV1 (26). The WPRE has the potential to provide a mechanism for their expression and, in so doing, to furnish a generic solution to the problem of expressing these genes from BHV1 vectors.…”

“…One strategy used to cope with this problem in the past has been to chemically synthesize a new version of the gene in which the defect is relieved if not eliminated. This strategy was used successfully to express the G protein of bovine respiratory syncytial virus (BRSV) and the E2 protein of type 1 BVDV (C86 strain) in BHV1 recombinants (26,50). With respect to the BVDV E2 protein, however, it should be noted that the unmodified, genomic form of the E2 open reading frame (ORF) from type 1 (NADL strain) BVDV has been successfully expressed in a BHV1 vector (27).…”

Recombinant Bovine Adenovirus Type 3 Expressing Bovine Viral Diarrhea Virus Glycoprotein E2 Induces an Immune Response in Cotton Rats

“…However, insertion of an intron upstream of E2ori did not alter the expression of the protein (data not shown), although a similar approach was successful in obtaining desirable expression of bovine corona virus E3 glycoprotein in the E3 region of BAV-3 (Reddy et al, 2000). Alternatively, it is possible that the transcripts are unstable in the nucleus (Kühnle et al, 1998). To enhance the expression of E2 protein, two different approaches were used.…”

“…This suggests that the presence of internal splice sites affects the proper processing of the E2specific transcripts. Previously, a similar approach was used successfully to express the BRSV G and BVDV E2 antigen in BHV-1 (Kühnle et al, 1998;Schmitt et al, 1999).…”

Antigen delivery systems for veterinary vaccine development

“…Further developments with BHV-1 as a vector used genomic Higher antibody titres and enhancement of frequency of IFN-gamma producing PBMCs [79] integration of expression cassettes coding for structural proteins of bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV), PRV, and Cryptosporidium parvum, a parasite which causes cryptosporidiosis, a zoonotic disease [85][86][87][88][89][90][91][92][93]. Integration of the expression cassettes into the BHV-1 genome resulted in replacement or inactivation of pathogenicity-associated genes like the thymidine kinase gene [88][89][90][91][92] or genes encoding glycoproteins gC [92], gG [92,93], gE [86], or gI [85,87]. Analyses of the immunogenic properties of the BHV-1 recombinants expressing PRV glycoproteins in mice revealed induction of a protective immune response against a lethal challenge with PRV.…”