Reconstitution of Semliki forest virus membrane (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1976
After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95.104 and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as "rosette"-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.
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.
pH-Dependent Fusion between the Semliki Forest Virus Membrane and Liposomes
Proceedings of The National Academy of Sciences, 1980
Semliki Forest virus was mixed with liposomes containing phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol. When the pH of the mixture was dropped to 6 or below, rapid fusion between the membranes of the virus and the liposomes occurred, resulting in the transfer of viral nucleocapsids into the liposomes. Fusion was demonstrated biochemically by trapping RNase or trypsin within the liposomes. Trapped RNase digested the viral RNA into acidsoluble form, providing a simple quantitative assay for fusion. Trapped trypsin digested the viral capsid protein. Fusion was also demonstrated by electron microscopy as the formation of large vesicles containing viral glycoproteins on the surface and nucleocapsids inside. The efficiency of fusion was 91 4 6%. In addition to low pH, it required that the viral glycoproteins be intact. In the target liposomes, cholesterol (but none of the individual phospholipids) was essential. Divalent cations were not required. Our previous studies with tissue culture cells indicated that the final step in the penetration of the Semliki Forest virus genome into host cells might involve a fusion event between the membrane of lysosomally trapped viruses and the lysosomal membrane [Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. (1980)J. Cell Biol. 84, 404-4201. The data presented here are fully compatible with this hypothesis. Membrane fusion events in biological systems are specific and carefully regulated processes mediating important physiological events such as secretion, endocytosis, fertilization, muscle development, and intracellular transport (1). In these cases, two membrane-limited compartments fuse so that the membranes and the aqueous contents are combined without detectable leakage of the contents. The orientation of the proteins in the two membranes remains unchanged. The actual molecular mechanisms underlying the fusion reaction are obscure and there is a need for suitable model systems to study this process. A model system should fulfill the criteria of membrane fusion as outlined above and it should, in addition, possess high fusion activity and provide a simple quantitative assay. In this paper we describe the fusion of Semliki Forest virus (SFV) with liposomes composed of phospholipids and cholesterol which appears to fulfill these requirements.
Interactions of Semliki Forest Virus Spike Rosettes and Vesicles with Cultured Cells Glycoprotein
Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (proteinfree lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH-dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions.
Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells
The Journal of Cell Biology
Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (proteinfree lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH-dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions.
Reversible defect in the glycosylation of the membrane proteins of Semliki forest virus ts-1 mutant
Virology, 1981
Here we have characterized the oligosaccharide chains ofthe viral membrane proteins from chicken embryo fibroblasts infected with tsl mutant of Semliki Forest virus. When the infected cells were labeled with [:'H]mannose and maintained at 39", the radioactive oligosaccharides were exclusively of the high mannose type as evidenced by their sensitivity to endoglycosidase H and their high affinity to immobilized concanavalin A. When the infected cells were labeled at 39" followed by chase at 28" in the presence of cycloheximide, about 35% of the high mannose type oligosaccharides were converted to complex oligosaccharides as evidenced by their elution behavior from concanavalin A-Sepharose, resistance to endoglycosidase H, and sensitivity to mild acid hydrolysis. The rest of the oligosaccharides remained as high mannose type chains. The results suggest that at the restrictive temperature the viral membrane glycoproteins in Is-l infected cells are arrested in the rough endoplasmic reticulum, but start to be transported to the Golgi complex, once the cultures are shifted to the permissive temperature. ' To whom reprint requests should be addressed. Z GlcNAc, A'-acetylglucosamine; Man, mannose.
Penetration of semliki forest virus from acidic prelysosomal vacuoles
Cell, 1983
To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37% the genome. release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15"-20°C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes.'Nevertheless, at 20°C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH < 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.