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.
AkkinaR. K.,
ChambersT. M.,
LondoD. R.,
NayakD. P.1987; Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB, protein from cloned cDNA. Journal of Virology 61:2217–2224
BishopD. H. L.1986; Ambisense RNA genomes of arenaviruses and phleboviruses. In Advances in Virus Research vol 31 pp 1–51 Edited by
MaramoroschK.,
MurphyF. A.,
ShatkinA. J.
Orlando: Academic Press;
BishopD. H. L.1990; Bunyaviridae and their replication. Part I: Bunyaviridae. In Virology 2nd edn pp 1155–1173 Edited by
FieldsB. N.,
KnipeD. M.
New York: Raven Press;
BishopD. H. L.,
ShopeR. E.1979; Bunyaviridae. In Comprehensive Virology vol 14 pp 1–156 Edited by
Fraenkel-ConratH.,
WagnerR. R.
New York: Plenum Press;
BouloyM.1991; Bunyaviridae: genome organization and replication strategies. In Advances in Virus Research vol 40 pp 235–275 Edited by
MaramoroschK.,
MurphyF. A.,
ShatkinA. J.
Orlando: Academic Press;
BouloyM.,
VialatP.,
GirardM.,
PardigonN.1984; A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein. Journal of Virology 49:717–723
ChenS. Y.,
MatsuokaY.,
CompansR. W.1991; Golgi complex localization of the Punta Toro virus G2 protein requires its association with the G1 protein. Virology 183:351–365
DaviesG. E.,
StarkG. R.1970; Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proceedings of the National Academy of Sciences, U.S.A. 66:651–656
DesprèsP.,
GirardM.,
BouloyM.1991; Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells. Journal of General Virology 72:1331–1342
DieckmannC. L.,
TzagoloffA.1985; Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. Journal of Biological Chemistry 260:1513–1520
DunphyW. G.,
BrandsR.,
RothmanJ. E.1985; Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cistemae of the Golgi stack. Cell 40:463–472
EshitaY.,
BishopD. H. L.1984; The complete sequence of the M RNA of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein. Virology 37:227–240
FazakerleyJ. K.,
Gonzalez-ScaranoF.,
StricklerJ.,
Dietz-scholdB.,
KarushF.,
NathansonN.1988; Organization of the middle RNA segment of snowshoe hare bunyavirus. Virology 167:422–432
GerbaudS.,
VialatP.,
PardigonN.,
WychowskiC.,
GirardM.,
BouloyM.1987; The S segment of the Germiston virus RNA genome can code for three proteins. Virus Research 8:1–13
JarvisD. L.,
SummersM. D.1989; Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. Molecular and Cellular Biology 9214–223
KlenkH. D.,
RottR.1980; Cotranslational and posttranslational processing of viral glycoproteins. Current Topics in Microbiology and Immunology 90:19–48
LeesJ. F.,
PringleC. R.,
ElliottR. M.1986; Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of the structural features in the bunyavirus glycoprotein gene product. Virology 148:1–14
LudwigG. V.,
ChristensenB. M.,
YullT. M.,
SchultzK. T.1989; Enzyme processing of La Crosse virus glycoprotein G1: a bunyavirus-vector infection model. Virology 171:108–113
LudwigG. V.,
IsraelB. A.,
ChristensenB. M.,
YullT. M.,
SchultzK. T.1991; Role of La Crosse virus glycoprotein in attachment of virus to host cells. Virology 181:564–571
MadoffD. H.,
LenardJ.1982; A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of La Crosse virus. Cell 28:821–829
PardigonN.,
VialatP.,
GirardM.,
BouloyM.1982; Characterization of Germiston virus RNA: molecular weight and oligonucleotide fingerprints of the three RNA species. Annales de Virologie (Institut Pasteur) 133E:461–472
PardigonN.,
VialatP.,
GerbaudS.,
GirardM.,
BouloyM.1988; Nucleotide sequence of the M segment of Germiston virus: comparison of the M gene product of several bunyaviruses. Virus Research 11:73–85
PerssonR.,
PetterssonR. F.1991; Formation and intracellular transport of a heterodimeric viral spike protein complex. Journal of Cell Biology 112:257–266
SangerF.,
NicklenS.,
CoulsonA. R.1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74:5463–5467
SchawallerM.,
SmithG. E.,
SkehelJ. J.,
WileyD. C.1989; Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV. Virology 172:367–369
SchmaljohnC. S.,
PattersonJ. L.1990; Bunyaviridae and their replication. Part II: Replication of Bunyaviridae. In Virology 2nd edn pp 1175–1194 Edited by
FieldsB. N.,
KnipeD. M.
New York: Raven Press;
SchmaljohnC. S.,
HastyS. E.,
RasmussenL.,
DalrympleJ. M.1986; Hantaan virus replication: effects of monensin, tunicamycin and endoglycosidases on the structural glycoproteins. Journal of General Virology 67:707–717
SpindlerK. R.,
RosserD. S. E.,
BerkA. J.1984; Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli
. Journal of Virology 49:132–141
StanleyK. K.,
LuzioJ. P.1984; Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO Journal 3:1429–1434
StrousG. J. A. M.,
LodishH. F.1980; Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus. Cell 22:709–717
TalmonY.,
PrasadB. V. V.,
ClerxJ. P. M.,
WangG. J.,
ChinW.,
HewlettM. J.1987; Electron microscopy of vitrified hydrated La Crosse virus. Journal of Virology 61:2319–2321
WeissC. D.,
LevyJ. A.,
WhiteJ. M.1990; Oligomeric organization of gp120 on infectious human immunodeficiency virus type 1 particles. Journal of Virology 64:5674–5677
WhittM. A.,
BuonocoreL.,
PrehaudC.,
RoseJ. K.1991; Membrane fusion activity, oligomerization and assembly of the rabies virus glycoprotein. Virology 185:681–688
WinklerG.,
RandolphV. B.,
CleavesG. R.,
RyanT. E.,
StollarV.1988; Evidence that the mature form of the flavivirus non-structural protein NS1 is a dimer. Virology 162:187–196
ZiemieckiA.,
GaroffH.,
SimonsK.1980; Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell. Journal of General Virology 50:111–123