Figure 4.

Vector characterization of RRV-2A encoding the yCD2 transgene in U87-MG cells. (A) Replication kinetics of RRV-2A-yCD2 variants. Viral genomes isolated from the viral supernatant of infected U87-MG cells were quantified by RT-qPCR using primers spanning the viral envelope region. Infected cells were passaged every other day through 10 days post infection, and supernatants were collected at each indicated time point. Data shown represent one of three independent experiments. RRV-IRES-yCD2 vector was included as control. (B) Western blot analysis of viral proteins produced by U87-MG cells maximally infected with RRV-2A-yCD2 variants. Twenty micrograms of total protein lysates was loaded per well. Membranes were incubated with anti-yCD2 antibody, which detects the viral envelope-yCD2 polyprotein (Env-yCD2). Detection of GAPDH using the anti-GAPDH antibody was included as a loading control. Protein standards are indicated in kDa. (C) LD₅₀ of 5-FC-mediated killing of maximally infected U87-MG cells with RRV-2A-yCD2 variants. Cells were cultured in the presence of 5-FC in different concentrations for 7 days. Naïve cells were included as a control for 5-FC cytotoxicity at each concentration. Cell viability was quantified by using MTS assay at 7 days post 5-FC addition, and the percentage of cell survival was calculated relative to RRV-infected cells not treated with5-FC. The data set represents one of the three independent experiments. Error bars indicate the standard deviation of the data set. Numbers in parentheses indicate LD₅₀ values. Statistical significance was determined by two-way ANOVA. The p-value for P2A-yCD2 versus gP2A-yCD2 is <0.0079; the p-value for T2A-yCD2 vs. gT2A-yCD2 is <0.0001.