Propagation, Purification, and In Vivo Testing of Oncolytic Vesicular Stomatitis Virus Strains
Oncolytic viruses are self-amplifying therapeutics that specifically replicate in and kill cancer cells. We have previously shown that vesicular stomatitis virus (VSV) can be used as an oncolytic virus. A strain of VSV harboring a mutation in the M protei
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ction Vesicular stomatitis virus (VSV) is a small negative-strand RNA virus belonging to the Rhabdovirus family. The VSV genome (approximately 11 kbp) encodes five distinct genes referred to as the nucleocapsid (N), polymerase proteins (P and L), the matrix protein (M), and the surface glycoprotein (G). The VSV virion is bullet-shaped and consists of viral RNA wrapped tightly within the ribonucleocapsid (an array of N, P, L proteins) to which binds M protein. The virion becomes enveloped with G protein trimers
David H. Kirn et al. (eds.), Oncolytic Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 797, DOI 10.1007/978-1-61779-340-0_10, © Springer Science+Business Media, LLC 2012
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upon budding from the host cell, which is the main target of neutralizing antibodies generated against the virus (1). VSV has been explored as an oncolytic virus due in part to its rapid life cycle and its potent cytolytic activity. Being an RNA virus that replicates in the cytoplasm, VSV is unlikely to recombine with cellular DNA and cannot integrate the genome. Combined with low human exposure to the virus and the absence of preexisting immunity in the population, this makes VSV an attractive candidate for human cancer therapy (2). Interferon (IFN) signaling is thought to be defective in approximately 70–75% of all tumors (3). In light of this, we and others have explored VSV strains that exploit this tumor-specific defect to enhance tumor targeting (4, 5). One of these mutant strains is VSV$51 which harbors a mutation in the M protein at methionine 51. Whereas wild-type M protein can block the nuclear export of cellular antiviral genes and their subsequent expression, the M$51 protein cannot and so VSV$51 replication is quickly subdued in normal cells that have intact antiviral signaling pathways (4).
2. Materials 2.1. Cell Culture and Virus Propagation
1. HyQ High glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, Waltham, MA). 2. Solution of trypsin (0.25%) and ethylenediamine tetraacetic acid (EDTA) (1 mM) from Gibco/BRL. 3. ViCell (Beckman/Coulter) automated cell counter (alternately any standard hemocytometer can be used). 4. Phosphate buffer saline (PBS, HyClone). 5. 0.20 Mm Bottle top filtration unit (Millipore).
2.2. Virus Purification
1. OptiPrep 60% (w/v) (Sigma). 2. Tris stock solution (100 ml): Dissolve 12.1 g Tris base into 100 ml ddH2O. Filter and store at 4°C. 3. EDTA stock solution (100 ml): Dissolve 3.72 g EDTA Na2·2H2O into 100 ml ddH2O. Filter and store at 4°C. 4. NaCl stock solution (100 ml): Dissolve 5.84 g NaCl into 100 ml ddH2O. Filter and store at 4°C. 5. Solution B (100 ml): To 50 ml water, add 30 ml Tris stock solution and 3.0 ml EDTA stock solution. Adjust pH to 7.4 with 5 M HCl, and adjust volume to 100 ml with ddH2O. Filter and store at 4°C.
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6. Solution C (100 ml): To 50 ml water, add 10 ml NaCl stock solution, 5.0 ml Tris stock
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