Development and Applications of VSV Vectors Based on Cell Tropism

Tani, Hideki; Morikawa, Shigeru; Matsuura, Yoshiharu

Seeded or recombinant VSVs in which the G gene is replaced by a foreign reporter gene such as a fluorescent reporter protein (green fluorescent protein, GFP; red fluorescent protein, RFP; and so on), luciferase, or SEAP or each viral envelope gene were generated as described below. Either 293T or BHK cells were grown to 90% confluence on 35-mm tissue culture plates. The cells were infected with a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase (vTF7-3) at a multiplicity of infection (MOI) of 5. After incubation at room temperature for 1 h, the cells were transfected with helper plasmids, pBS-N, pBS-P, pBS-L, and pBS-G, and template plasmids, pVSVΔG-GFP (RFP), pVSVΔG-Luci, pVSVΔG-SEAP, or pVSVΔG-Env using a cationic liposome reagent. After 4 h, the supernatants were Table 1 | Application studies of pseudotype and recombinant VSV. Takada replaced with 10% FBS DMEM, and cells were incubated at 37˚C for 48 h. The supernatants were then filtered through a 0.22-μmpore-size filter to remove vaccinia virus and were applied to 293T or BHK cells that had been transfected with pCAGVSVG 24 h previously. If BHK cells constitutively expressing the bacteriophage T7 RNA polymerase (BHKT7) were utilized, the cells were only transfected with helper plasmids, pIRES-N, pIRES-P, pIRES-L, pIRES-G, and template plasmids using a cationic liposome reagent without the vaccinia virus infection. Recovery of the virus was assessed by examining the cells for the cytopathic effects that are typical of a VSV infection after 24 h. Stock of * G-complemented viruses, i.e.,VSVΔG virus or recombinant viruses transiently bearing VSV G protein on the virion surface, were grown from the single plaque on BHK cells transfected with pCAGVSVG and then stored at −80˚C. The infectious titers of the recovered viruses were determined by a plaque assay. To generate pseudotype virus, 293T, BHK, or some other type of cells that exhibit a high competency of transfection were transfected with a plasmid expressing the envelope protein using a cationic liposome reagent. After 24 h of incubation at 37˚C, cells were infected at an MOI of 0.5 with the * G-VSVΔG-Luci and * G-VSVΔG-SEAP, or 5 with * G-VSVΔG-GFP (RFP). The virus was adsorbed for 2 h at 37˚C and then extensively washed four times or more with serum-free DMEM.