Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors.

Abstract

The major glycoprotein, G, of human respiratory syncytial (RS) virus is a M, 84,000-90,000 species that has about 60% of its mass contributed by carbohydrate, most of which is in the form of O-linked oligosaccharides. The G protein contains neither a hydrophobic N-terminal signal sequence nor a hydrophobic C-terminal anchor region. Instead, its amino acid sequence reveals only one region with significant hydrophobic character, which is between residues 38 and 66. In order to study the synthesis, processing, and functions of this unusual viral glycoprotein, full-length cDNA copies of the G protein mRNA were inserted into the DNA genome of vaccinia virus (VV) in a position that was adjacent to a strong W promoter and within the W gene for thymidine kinase (TK). The resulting TK-recombinant viruses were selected, plaque-purified, and characterized by Southern blot analysis of restriction enzyme digests of the viral DNA. Recombinant RNA transcripts that contained both G-specific and W-specific sequences accumulated in cells infected with recombinant viruses having the G protein gene in the positive orientation. The translation product of these transcripts in infected cells was a Mr 84,000-90,000 glycoprotein that was indistinguishable from authentic RS virus G protein. It could be detected in cell lysates after metabolic labeling with [3H]glucosamine and was immunoprecipitated by anti-RSvirus antiserum. Immunofluorescence studies showed that the G protein accumulated intracellularly with the perinuclear distribution that is characteristic of newly synthesized glycoproteins. Furthermore, the protein was also clearly detectable on the surface of recombinant-infected cells, showing that it was transported to and inserted into the plasma membrane.Human respiratory syncytial (RS) virus is an important human pathogen that causes severe lower respiratory tract disease, particularly among young children (1). The related virus, bovine RS virus, causes severe respiratory disease in cattle (2). No effective vaccine has been developed for either virus; moreover, natural infection with the human virus frequently fails to confer immunity. Thus, recurrent epidemics occur during which human RS virus is a major cause of hospitalization of children with lower respiratory tract disease (1, 2).Progress in understanding the molecular biology of RS virus and the role of individual proteins in infection and immunity has been slow because the virus replicates poorly in cell culture and is extremely labile, which makes it difficult to obtain adequate quantities of the virus or viral proteins for experimentation. Recently, however, as a result of cDNA cloning, substantial progress has been made in our knowledge of the molecular biology of human RS virus (3-5). Classified as genus Pneumovirus within the Paramyxoviridae family (6), human RS virus has a genome of single-stranded, negativesense RNA of about Mr 5,600,000 that encodes 10 unique viral proteins (4, 7-12). In order to understand the pathogenesis of human RS virus infection an...

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