Figure 3.

RNA and protein expression from U87-MG cells infected with RRV containing various numbers of A's in the A bulge. (A) Schematic diagram of cellular viral RNA isoforms. The env2 and yCD2 primer sets, which recognize both unspliced and spliced viral RNA in the env and the yCD2 region, respectively, were used to measure the level of cellular viral RNA by qRT-PCR. Filled triangles, env2 primer set; open triangles, yCD2 primer set. (B) Immunoblot of yCD2 and the GAPDH protein. Twenty micrograms of cell lysate of maximally infected U87-MG cells from one round of infection was loaded to each lane, and equivalent loading and blotting efficiency controlled for by detection of the ubiquitous marker GAPDH. NC, negative control from naïve cell lysates. (C) Histograms represent the RNA and protein expression levels relative to the yCD2-6A vector. RNA expression was performed in triplicates and the relative level of viral RNA from each vector was calculated using 2^−ΔΔ(Ct) method with respect to the vector containing the 6 A's. Protein expression levels of yCD2 were calculated relative to vector containing the 6 A's. (D) Stability of proviral DNA of IRES-yCD2 cassette in RRV-IRES-yCD2 variants from one round of infection showed no detection of deletion mutants. Arrow indicates the expected 1.2 kb PCR product containing the IRES-yCD2 cassette. (E) Cell-based enzymatic activity of yCD2 in infected U87-MG cells was measured by HPLC to detect the amount of 5-FU. The 5-FU peak area of each vector is plotted relative to yCD2-6A vector that is set to 1. Data shown in (B–D) are from representative examples of two or three independent experiments. Data shown in (E) represent mean ± SD relative to 6A from three independent experiments.