Rapid purification of extracellular and intracellular Moloney murine leukemia virus

Abstract

The present study demonstrates the advantages of a combination of concentration by polyethylene glycol-6000 and Sepharose Cl-4B chromatography as a rapid procedure for retroviruses purification. This procedure can be completed within 3 hours, providing a high degree of virus purification with minimal damage to its structural and biological properties. Using transmission electron microscopy we observed many intracellular type-C virions in cytoplasmic vacuoles of 3T3/NIH cells chronically infected with Moloney murine leukemia virus. There intracellular virions could be isolated from postmitochondrial cytoplasmic fractions prepared from the infected cells by a procedure which minimized its contamination by extracellular free or membrane-bound virions. SDS-polyacrylamide gel electrophoresis showed that the intracellular and extracellular virions contained similar protein composition.

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

“…At small scale viruses can be concentrated and purified via sucrose density centrifugation. This results in low production rates and low yields but high purity virus dispersions (Aboud et al, 1982;Aranha, 2001). At large scale, virus membrane filtration represents a beneficial trade-off.…”

Enhanced retroviral gene delivery in ultrasonic standing wave fields

“…25,26 This is because the association of gp70 (binding to cellular receptor) and p15E (fusion with plasma membrane) subunits of type C retroviral envelope protein is a labile disulfide linkage, which is not robust under shearing forces. 27,28 Owing to the loss of gp70 subunit (or SU domain), the retrovirus cannot enter the target cell via receptor-mediated endocytosis. Since VSV-G consisting of a single polypeptide chain rather than two disulfide-linked chains has been demonstrated to be sturdy under the shearing force used to concentrate retrovirus by ultracentrifugation, 22,29 VSV-G pseudotyped retrovirus was thereby selected for its potency of retaining infectivity under USWF irradiation.…”

Technical Considerations for the Generation of Novel Pseudotyped Viruses

“…Optimization of incubation time and molecular weight and concentration of PEG used will all be required, however numerous commercial kits are available with specific protocols. An optimized method for MLV uses 8.5% (w/v) PEG 6000 for 90 minutes at 4°C followed by collection of the precipitate by centrifugation at 7,000 x g for 10 minutes [106,107].…”