Reversible defect in the glycosylation of the membrane proteins of Semliki forest virus ts-1 mutant (original) (raw)
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Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell
Journal of General …, 1980
In Semliki Forest virus (SFV)-infected cells, all structural proteins are translated from a 26S mRNA using a single initiation site. The capsid protein which is made first is released into the cytoplasm whereas the two membrane proteins, p62 (the precursor for E2 and E3) and El, are inserted into the rough endoplasmic reticulum membrane. Based on gradient centrifugation and cross-linking studies, it can be seen that the p62 and Et polypeptides form a compleximmediately after synthesis and migrate to the plasma membrane in the form of a p62-EI complex. The processing of p62 to E2 and E3 is first seen 25 to 30 min after a IO min pulse of radioactive amino acids. This cleavage can be inhibited by addition of antisera specific for EI and E3, thus supporting the view that, as in the case of the related Sindbis virus, this cleavage occurs on the external face of the plasma membrane. Proteolytic digestion of crude vesicle preparations derived from plasma membranes, combined with peptide mapping, indicate that the carboxy-terminal end of E2 spans the cell plasma membrane, there being a portion of mol. wt about 3ooo located towards the cytosol.
Sindbis virus glycoproteins: effect of the host cell on the oligosaccharides
Journal of Virology
Sindbis virus was grown in four different host cells and the carbohydrate portions of the glycoproteins were analyzed. Sindbis virus grown in BHK-21 cells has more sialic acid and galactose than Sindbis virus grown in chicken embryo cells. In other respects the carbohydrates from virus grown in these two hosts are very similar. Sindbis virus grown either in chick cells transformed by Rous sarcoma virus or in BHK cells transformed by polyoma virus was also examined. In comparisons of virus from normal and transformed cells, differences in the amount of sialic acid were observed; but otherwise the carbohydrate structures appeared basically similar. The growth conditions used for the host cell also affected the degree of completion of the carbohydrate chains of the viral glycoproteins.
Journal of virology, 1981
When Semliki Forest virus temperature-sensitive mutant ts-3 was grown at the restrictive temperature an aberrant nascent cleavage of the 130,000-dalton structural polyprotein took place relatively frequently. This cleavage yielded an abnormal 86,000-dalton fusion protein (p86) consisting of the amino-terminal capsid protein linked to the amino acid sequences of envelope protein p62 (a precursor of E3 and E2). The other cleavage product was the carboxy-terminal envelope protein E1. p86 was not glycosylated and was sensitive to the action of protease in the microsomal fraction, whereas E1 was glycosylated and protected from proteases, indicating that it had been segregated into the cysternal side of the microsomal vesicles. All attempts to show the E1 protein at the cell surface have failed so far, suggesting that it remains associated with intracellular membranes. When ts-3-infected cells labeled at the restrictive temperature were shifted to the permissive temperature the only label...
Characterization of the Oligosaccharides of Inkoo Virus Envelope Glycoproteins
Journal of General Virology, 1982
Methionine labelled the two envelope glycoproteins G1 (Mr = 125000) and G2 (Mr = 35000), as well as the nucleocapsid protein N (Mr = 25000). Only G1 and G2 were labelled with the sugar precursor. The [3H]mannoselabelled virus was solubilized with detergent and digested with Pronase. The structure of the labelled glycopeptides originating from the mixture of G 1 and G2 was studied by degrading the glycans stepwise with specific exo-and endoglycosidases, and by analysing the products by both gel and paper chromatography, as well as lectin-affinity chromatography. Three classes of N-glycosidic glycans were found: complex glycans with the monosaccharide sequence (NeuNAc~GalpGlcNac/~)_>2 (Man)3 (GIcNAc)2 (occurrence of fucose was not studied), high mannose-type chains with the average structure (Man)4_6 (GIcNAc)2, and endoglycosidase H-resistant small glycans which were partly susceptible to mannosidase. These latter types of oligosaccharide chains are a novel finding among virus glycoproteins. The relative ratio of the three types of oligosaccharide chains was roughly 4.6:1 : 1 respectively. The G 1 glycoprotein carried most of the sugar chains, since it contained 85 ~o of the [3H]mannose label. The results are discussed in relation to the site of virus maturation at smooth-surfaced vesicles in the Golgi region. 0022-1317/82/0000-5123 $02.00 © 1982 SGM Downloaded from www.microbiologyresearch.org by IP: 54.145.26.59 On: Sat, 02 Apr 2016 11:37:25 426 M. PESONEN AND OTHERS accumulating due to the intracellular maturation site of the virus (Pesonen et al. , 1982). In the present study, the same three categories of protein-bound oligosaccharides were found in Inkoo virus. were infected with Inkoo virus, strain KN3641 , at an m.o.i of 5. The prototype strain virus was plaquepurified three times successively and a stock prepared using about 0-05 p.f.u./cell. The stock virus had a titre of l0 s p.f.u./ml.
Journal of Virology, 1979
Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N -acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two res...
Reconstitution of Semliki forest virus membrane
Journal of Cell Biology, 1977
The spike glycoproteins of the Semliki forest virus membrane have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis method. The detergent used was beta-D-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by hydrophobic peptide segments, as in the viral membrane. Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation.
Journal of Virology, 1989
The synthesis of the extensively 0-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, IdID, which has a defect in protein 0 glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or 0-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant IdlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or 0-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the 0-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of 0-linked sugar addition, confirming that 0-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of 0 glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N-and 0-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either 0 glycosylation or N glycosylation. However, cell surface expression of G lacking both N-and 0-linked oligosaccharides was severely depressed.
Virology, 2004
Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G N (N-terminal) and G C (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G N and G C . The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G C showed strong binding with five mannose-binding lectins, four Nacetyllactosamine-binding lectins and one fucose-binding lectin. G N was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G C , with three of the five mannose-binding lectins. The N-acetyllactosamine-and fucosespecific lectins did not bind to either molecular mass form of G N . None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G C or G N . Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G C to high mannose-and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G N . These results indicate the presence of N-linked oligosaccharides of high mannoseand complex-type on G C and possibly high mannose-type on G N . Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G C is likely to be more heavily N-glycosylated than G N . No evidence was observed for the presence of O-linked oligosaccharides on G N or G C .