Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins.
We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.
Gerbaud
S
S
Unité de Virologie Moléculaire, CNRS URA 545, Paris, France.
Pardigon
N
N
Vialat
P
P
Bouloy
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000918:Antibody Specificity; D002042:Bunyamwera virus / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014759:Viral Envelope Proteins / Q000737:chemistry*; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D006023:Glycoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; D006026:Glycoside Hydrolases