FIG. 6.

Role of NS gene in MVM infection of human astrocytic tumor cells. (A) Southern blot analysis of MVMi and MVMi-NSp viruses DNA synthesis in U373 (lanes 1 and 2) and U87 (lanes 3 and 4) cells. Samples from 2 × 10⁵ cells infected at the indicated MOI and proceeded at 2 hpi (lanes 1 and 3) or 24 hpi (lanes 2 and 4) were loaded per gel slot, and filters were hybridized with a full-length MVM probe labeled to high specific activity. Exposure was for 48 h (MOI of 0.1) or 8 h (MOI of 5) with intensifying screen at −70°C. Markers are as described for previous figures. (B) Production of infectious MVM virus in culture of human nervous cells. Cells were scraped into the medium at the indicated postinfection times, and the total MVMi or MVMi-NSp virus produced in the cultures was determined by a plaque assay in NB3234K cells. (C) IF analysis of MVMi and MVMi-NSp virus progression in culture. Monolayers of human cells growing on coverslips were inoculated at an MOI of 0.01 PFU/cell and analyzed by IF for VP protein synthesis at the indicated times postinfection. The plots show a quantitative average of IF⁺ cells for VP nuclear staining from four independent experiments. Values are represented as the normalized fold increase with respect to the number of IF⁺ nuclei determined at 24 hpi.