scholarly journals Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization.

1985 ◽  
Vol 101 (2) ◽  
pp. 460-469 ◽  
Author(s):  
C A Gabel ◽  
J E Bergmann
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Complex Type ◽  
Major Class ◽  
Vesicular Stomatitis ◽  
High Mannose

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.

scholarly journals Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

1987 ◽  
Vol 104 (3) ◽  
pp. 749-760 ◽  
Author(s):  
W E Balch ◽  
K R Wagner ◽  
D S Keller
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Free System ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

scholarly journals Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport.

1989 ◽  
Vol 109 (5) ◽  
pp. 2057-2065 ◽  
Author(s):  
D Mack ◽  
B Kluxen ◽  
J Kruppa
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Protein Domain ◽  
Complex Type ◽  
Carbonyl Cyanide ◽  
Vesicular Stomatitis ◽  
High Mannose

G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins

1988 ◽  
Vol 8 (7) ◽  
pp. 2869-2874
Author(s):  
J L Guan ◽  
A Ruusala ◽  
H Cao ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Cytoplasmic Domain ◽  
Secretory Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

scholarly journals Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

1986 ◽  
Vol 102 (6) ◽  
pp. 2147-2157 ◽  
Author(s):  
L Puddington ◽  
C E Machamer ◽  
J K Rose
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Heavy Chain ◽  
Cytoplasmic Domain ◽  
Integral Membrane Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Hybrid Proteins ◽  
Vesicular Stomatitis ◽  
Immunoglobulin Mu

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

scholarly journals Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

1988 ◽  
Vol 8 (7) ◽  
pp. 2869-2874 ◽  
Author(s):  
J L Guan ◽  
A Ruusala ◽  
H Cao ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Cytoplasmic Domain ◽  
Secretory Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

scholarly journals Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein.

1983 ◽  
Vol 97 (5) ◽  
pp. 1381-1388 ◽  
Author(s):  
R Z Florkiewicz ◽  
A Smith ◽  
J E Bergmann ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Rough Endoplasmic Reticulum ◽  
Mouse Cell ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Truncated Form ◽  
Vesicular Stomatitis

We have characterized two stable transformed mouse cell lines (CG1 and CTG1) that express either the normal vesicular stomatitis virus glycoprotein (G) or a truncated form of the G protein (TG) that lacks the COOH-terminal anchor sequences and is secreted from the cells. These cell lines were obtained using a hybrid vector consisting of the transforming DNA fragment of bovine papilloma virus linked to a segment of the SV40 expression vector pSV2 containing cloned cDNA encoding either the normal or truncated form of the vesicular stomatitis virus G protein. Using indirect immunofluorescence we have found that greater than 95% of the cells in each line express the G protein(s), although the level of expression within the population is variable. The normal G protein expressed in these cells obtains its complex oligosaccharides in less than 30 min and is transported to the cell surface. In contrast, the TG protein obtains its complex oligosaccharides with a half-time of about 2.5 h. Immunofluorescence data show an apparent concentration of the TG protein in the rough endoplasmic reticulum. These data together suggest that transfer of this anchorless protein from the rough endoplasmic reticulum to the Golgi apparatus is the rate-limiting step in its secretion. We observed, in addition to normal G protein, two smaller G-related proteins produced in the CG1 cell line. We suggest that these proteins could result from aberrant splicing from sites within the G mRNA sequence to the downstream acceptor in the pSV2 vector.

scholarly journals Tubulovesicular Structures within Vesicular Stomatitis Virus G Protein-Pseudotyped Lentiviral Vector Preparations Carry DNA and Stimulate Antiviral Responses via Toll-Like Receptor 9

2006 ◽  
Vol 81 (2) ◽  
pp. 539-547 ◽  
Author(s):  
Andreas Pichlmair ◽  
Sandra S. Diebold ◽  
Stephen Gschmeissner ◽  
Yasuhiro Takeuchi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Gene Therapy ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Lentiviral Vector ◽  
Virus Production ◽  
Toll Like Receptor ◽  
Cellular Origin ◽  
Antiviral Responses ◽  
Vesicular Stomatitis

ABSTRACT Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.

Sign in / Sign up

Export Citation Format

Share Document