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Title: Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus

Abstract

A virus pseudotyped with glycoprotein of vesicular stomatitis virus (VSV-G) can enter various cell types at a relatively high titer. We observed that the amount of viral antigen from VSV-G pseudotyped human immunodeficiency virus type 1 (HIV-1) producing cells was much higher than that from their non-pseudotyped counterparts. This enhanced viral antigen production was not observed when we used HIV-1 pol mutant, viral enzyme inhibitors, HIV Env protein, or VSV-G fusion defective mutants. The transfection experiment using GFP-expressing virus showed time-dependent expansion of GFP-positive cells and viral DNA integration. These results suggested that the increase in viral antigen yield was caused by the release of a progeny virus following retro-transduction by the pseudotyped virus of the cells within the transfected cell culture. The infectivity as well as the amount of VSV-G on virus particles per unit of viral antigen was significantly different before and after the onset of the yield enhancement. This suggests that results of infection assays of the virus pseudotyped with VSV-G may be affected by the occurrence of such enhancement. This means that, while pseudotyping with VSV-G is a simple and effective method, this procedure should be carefully considered when the virus is produced for infectivity assays.

Authors:
 [1];  [1];  [2]
  1. Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871 (Japan)
  2. Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871 (Japan). E-mail: sakuragi@biken.osaka-u.ac.jp
Publication Date:
OSTI Identifier:
20977026
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 362; Journal Issue: 1; Other Information: DOI: 10.1016/j.virol.2006.12.030; PII: S0042-6822(06)00939-1; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AIDS VIRUS; ANTIGENS; CELL CULTURES; DNA; ENZYME INHIBITORS; GLYCOPROTEINS; INFECTIVITY; MUTANTS; TIME DEPENDENCE

Citation Formats

Ohishi, Masahisa, Shioda, Tatsuo, and Sakuragi, Jun-ichi. Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus. United States: N. p., 2007. Web. doi:10.1016/j.virol.2006.12.030.
Ohishi, Masahisa, Shioda, Tatsuo, & Sakuragi, Jun-ichi. Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus. United States. doi:10.1016/j.virol.2006.12.030.
Ohishi, Masahisa, Shioda, Tatsuo, and Sakuragi, Jun-ichi. Fri . "Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus". United States. doi:10.1016/j.virol.2006.12.030.
@article{osti_20977026,
title = {Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus},
author = {Ohishi, Masahisa and Shioda, Tatsuo and Sakuragi, Jun-ichi},
abstractNote = {A virus pseudotyped with glycoprotein of vesicular stomatitis virus (VSV-G) can enter various cell types at a relatively high titer. We observed that the amount of viral antigen from VSV-G pseudotyped human immunodeficiency virus type 1 (HIV-1) producing cells was much higher than that from their non-pseudotyped counterparts. This enhanced viral antigen production was not observed when we used HIV-1 pol mutant, viral enzyme inhibitors, HIV Env protein, or VSV-G fusion defective mutants. The transfection experiment using GFP-expressing virus showed time-dependent expansion of GFP-positive cells and viral DNA integration. These results suggested that the increase in viral antigen yield was caused by the release of a progeny virus following retro-transduction by the pseudotyped virus of the cells within the transfected cell culture. The infectivity as well as the amount of VSV-G on virus particles per unit of viral antigen was significantly different before and after the onset of the yield enhancement. This suggests that results of infection assays of the virus pseudotyped with VSV-G may be affected by the occurrence of such enhancement. This means that, while pseudotyping with VSV-G is a simple and effective method, this procedure should be carefully considered when the virus is produced for infectivity assays.},
doi = {10.1016/j.virol.2006.12.030},
journal = {Virology},
number = 1,
volume = 362,
place = {United States},
year = {Fri May 25 00:00:00 EDT 2007},
month = {Fri May 25 00:00:00 EDT 2007}
}
  • The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three aminomore » acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.« less
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  • The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganizationmore » of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.« less
  • Cells infected with temperature-sensitive (ts) mutants of complementation group V of vesicular stomatitis virus (VSV) give an enhanced yield at nonpermissive temperature when co-infected or superinfected with uv- irradiated virus. Virions produced in these mixed infections are temperature sensitive and do not complement ts V45. Rescue of group V mutants is multiplicity dependent. It can occur in the presence of cycloheximide; kinetics of rescue are similar in the absence or in the presence of the drug. Rescue is due to nongenetic complementation and is interpreted as a trigger effect on maturation of a small quantity of biologically active protein Vmore » molecules provided by uv-irradiated virus. These results are confirmed by rescue of ts V45 by uv-irradiated, defective, interfering T particles. (auth)« less