Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Type of study
Language
Publication year range
1.
J Virol ; 71(6): 4717-27, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9151865

ABSTRACT

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.


Subject(s)
Golgi Apparatus/microbiology , Membrane Glycoproteins/metabolism , Uukuniemi virus/growth & development , Viral Proteins/metabolism , Amino Acid Sequence , Biological Transport , CD4 Antigens/metabolism , Cell Compartmentation , Cysteine/chemistry , Cytoplasm/chemistry , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , Membrane Glycoproteins/chemistry , Molecular Sequence Data , Palmitates/metabolism , Protein Processing, Post-Translational , Recombinant Fusion Proteins , Sequence Deletion , Structure-Activity Relationship , Uukuniemi virus/genetics , Viral Envelope Proteins/chemistry , Viral Proteins/chemistry
2.
Virology ; 211(1): 241-50, 1995 Aug 01.
Article in English | MEDLINE | ID: mdl-7645217

ABSTRACT

We have studied the interactions of the G1 and G2 membrane glycoproteins of Uukuniemi virus, a bunyavirus, in virus particles and in Triton X-100-solubilized virus. The G1 glycoprotein in intact virus or in Triton solution could be oxidized into a covalent homodimer using Cu2+ ion as a catalyst. Immunoprecipitations of the glycoproteins from Triton-solubilized virus lysates showed that G1 and G2 do not form a stable heterodimeric or heterooligomeric complex. The oligomeric association of G1 and G2 was further analyzed using centrifugation in sucrose gradients in the presence of Triton X-100. The results indicate that G1 exists as a Triton-resistant pH-insensitive homodimer. This is in contrast to the behavior of G2, which exists as a homodimer and partially as a monomer at pH 6.4 or above and is dissociated completely into a monomer at pH 6.0 or below. The threshold for the dimer-monomer shift of G2 is between pH 6.2 and pH 6.0. Electron microscopy studies show that the surface structure of the virus particle undergoes a pH-dependent change. Studies on the kinetics of virus entry suggest that pH below 6.2 is necessary for the penetration of Uukuniemi virus.


Subject(s)
Uukuniemi virus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Animals , Cell Line , Cells, Cultured , Centrifugation, Density Gradient , Chick Embryo , Copper/pharmacology , Cricetinae , Fibroblasts , Kidney , Macromolecular Substances , Methionine/metabolism , Microscopy, Electron , Molecular Weight , Octoxynol , Oxidation-Reduction , Solubility , Sulfur Radioisotopes , Uukuniemi virus/growth & development , Uukuniemi virus/ultrastructure , Viral Envelope Proteins/ultrastructure
SELECTION OF CITATIONS
SEARCH DETAIL
...