Studies on the size, chemical composition, and partial sequence of the neuraminidase (NA) from type A influenza viruses show that the N-terminal region of the NA is not processed and serves to anchor the NA in the viral membrane

Blok, J.; Air, Gillian M.; Laver, W.G.; Ward, Colin W.; Lilley, Glen G.; Woods, E. F.; Roxburgh, C. M.; Inglis, A.S.

doi:10.1016/0042-6822(82)90069-1

Cited by 126 publications

(38 citation statements)

References 41 publications

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“…Serine and threonine residues, which provide the sites for O-glycosylation, constitute 30.6% of the protein. In addition, four potential sites for the attachment of N-linked oligosaccharides have been identified, and at least some of them appear to be glycosylated (11,16).The unusual structure of this glycoprotein raises many fundamental questions concerning the mechanisms of its synthesis, membrane insertion and anchoring, N-and 0-glycosylation, and transport to the plasma membrane. To address these questions and to examine the role of the G protein in the immune response to RS virus infection, we have inserted full-length cDNA copies of the G protein into eukaryotic expression vectors derived from vaccinia virus (VV) (17,18).…”

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“…If this is a transmembrane region, its location suggests that the G protein may be oriented with its C terminus on the external side of the membrane, like the influenza virus neuraminidase (14,15). However, unlike the latter protein, the mature RS virus G protein contains more than 60% of its mass as carbohydrate, most of which is comprised of O-linked oligosaccharide chains (11,16). Serine and threonine residues, which provide the sites for O-glycosylation, constitute 30.6% of the protein.…”

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

Ball¹,

Young²,

Anderson³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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

Ball¹,

Young²,

Anderson³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The mature NA glycoprotein is anchored in the virion envelope via its NH2 terminus, in contrast to the HA glycoprotein, which is anchored by the COOH terminus (5,9,13). Because of this difference in membrane orientation, it was of interest to investigate the site of surface expression of NA on influenza virus-infected polarized cells.…”

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Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

Jones¹,

Compans²,

Davis³

et al. 1985

Mol. Cell. Biol.

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We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold-or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that (i) the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and (ii) the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

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“…Like most membrane proteins, HA contains an Nterminal signal sequence which is cleaved off after translocation across the endoplasmic reticulum membrane, and a C-terminal sequence for anchoring the molecule in the plasma membrane. However, NA is oriented in the membrane in the opposite way to HA; the membrane anchor sequence is located at the N terminus and a signal sequence is not removed (Fields et al, 1981 ;Blok et al, 1982). Immunofluorescence analysis and a haemadsorption assay showed the presence of both proteins at the surface of AcUW3HANA-infected Sf cells, demonstrating that both types of glycoprotein can be expressed in insect cells and incorporated into the plasma membrane.…”

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confidence: 95%

A Baculovirus Dual Expression Vector Derived from the Autographa Californica Nuclear Polyhedrosis Virus Polyhedrin and p10 Promoters: Co-expression of Two Influenza Virus Genes in Insect Cells

Weyer¹,

Possee²

1991

Journal of General Virology

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Studies on the size, chemical composition, and partial sequence of the neuraminidase (NA) from type A influenza viruses show that the N-terminal region of the NA is not processed and serves to anchor the NA in the viral membrane

Cited by 126 publications

References 41 publications

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

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

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

A Baculovirus Dual Expression Vector Derived from the Autographa Californica Nuclear Polyhedrosis Virus Polyhedrin and p10 Promoters: Co-expression of Two Influenza Virus Genes in Insect Cells

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