In Vitro Cell-Free Conversion of Noninfectious Moloney Retrovirus Particles to an Infectious Form by the Addition of the Vesicular Stomatitis Virus Surrogate Envelope G Protein
- Akihiro Abe † ,
- Shin-Tai Chen ‡ ,
- Atsushi Miyanohara, and
- Theodore Friedmann *
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Fig. 1.Intracellular and extracellular VSV-G in cell lines transfected with pCMV-G. Cells were grown in six-well plates and transfected with plasmid pCMV-G. VSV-G samples in the conditioned medium and cell lysate were examined by established Western blotting methods, using P5D4 monoclonal antibody to visualize VSV-G protein as described in Materials and Methods.
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Fig. 2.Analysis of virus particles by sucrose velocity gradient centrifugation. (A) Authentic pseudotyped LLRNL virus prepared by conventional methods from 293GP/LLRNL cells. (B) Untreatedgag-pol RNA particles and gag-pol RNA particles made infectious by exposure to VSV-G for which 0.5-ml volumes of conditioned medium of 293 cells transfected with pCMV-G and of 293GP/LLRNL cells were mixed and centrifuged at 24,000 rpm at 4°C in Beckman SW28 rotor for 90 min. Pellets were resuspended with 1 ml of PBS and loaded onto the sucrose gradients. (C) Western blot analysis of VSV-G in gradient fractions containing the media from 293 cells transfected with pCMV-G, authentic mature pseudotyped LLRNL virus, andgag-pol RNA particles from 293GP/LLRNL cells made infectious by treatment with VSV-G as described above. RLU, relative luciferase units.
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Fig. 3.Stability of VSV-G, gag-pol RNA, and authentic pseudotyped virus particles. (A) Stability in incubation at 37°C. VSV-G-containing conditioned medium from 293 cells transfected with pCMV-G and gag-pol RNA (GP/LLRNL)-containing conditioned medium from 293GP/LLRNL cells (0.5 ml of each) were independently incubated at 37°C for the indicated time. After that, they were mixed with equal volumes of the other fresh components, centrifuged at 14,000 rpm for 1 h, and used to infect BHK cells. Authentic pseudotyped virus was made by the conventional method and incubated at 37°C. The activity of each component was determined by luciferase assay and expressed as the percentage of fresh-sample activity. An identical experiment in which samples were incubated at 4°C instead of 37°C resulted in a similar profile (data not shown). (B) Stability during freeze-thaw. Each sample (0.5 ml) was subjected to freezing at −80°C for 15 min and thawing at 37°C for 5 min, and each sample was kept at 4°C until the last samples were prepared. Then VSV-G or gag-pol RNA particles were mixed with equal volumes of fresh other components, centrifuged at 14,000 rpm for 1 h, and subjected to infection. Each value represents the percentage of fresh-sample activity. Results are the mean ± standard error of three independent experiments.
- Copyright © 1998 American Society for Microbiology
