scholarly journals Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

1996 ◽  
Vol 93 (19) ◽  
pp. 10057-10062 ◽  
Author(s):  
S. T. Chen ◽  
A. Iida ◽  
L. Guo ◽  
T. Friedmann ◽  
J. K. Yee
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Retroviral Vectors ◽  
Packaging Cell ◽  
Vesicular Stomatitis ◽  
Tetracycline Inducible System

scholarly journals A New System for Stringent, High-Titer Vesicular Stomatitis Virus G Protein-Pseudotyped Retrovirus Vector Induction by Introduction of Cre Recombinase into Stable Prepackaging Cell Lines

1998 ◽  
Vol 72 (2) ◽  
pp. 1115-1121 ◽  
Author(s):  
Tohru Arai ◽  
Kazuyuki Matsumoto ◽  
Kanako Saitoh ◽  
Motoyasu Ui ◽  
Taiji Ito ◽  
...  
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
High Titer ◽  
Cre Recombinase ◽  
Transcriptional Unit ◽  
Fourfold Increase ◽  
Packaging Cell ◽  
Retrovirus Vector ◽  
Vesicular Stomatitis

ABSTRACT We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-polgenes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neor) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neor expression. By inserting an mRNA-destabilizing signal into the 3′ untranslated region of the Neor gene to reduce the amount of Neor transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding β-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 × 106 infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

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.

Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors

1998 ◽  
Vol 73 (1) ◽  
pp. 31-39 ◽  
Author(s):  
William C Manning ◽  
John E Murphy ◽  
Douglas J. Jolly ◽  
Steven J Mento ◽  
Robert O Ralston
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Retroviral Vectors ◽  
Murine Cytomegalovirus ◽  
Vesicular Stomatitis

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

1980 ◽  
Vol 33 (1) ◽  
pp. 411-422 ◽  
Author(s):  
F Wyers ◽  
C Richard-Molard ◽  
D Blondel ◽  
S Dezelee
Keyword(s):  
Drosophila Melanogaster ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Protein Deficiency ◽  
Virus Growth ◽  
Vesicular Stomatitis

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

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