FIG. 2.

Attenuated gene expression by recombinant MVs with the P and/or L genes of the Edmonston strain. (A) Confluent monolayers of various cell lines (Vero/hSLAM, CV1/hSLAM, HeLa/hSLAM, CHO/hSLAM, and B95a) and suspensions of nonadherent MT2 cells cultured in 24-well cluster plates were infected with 2.5 × 10³ PFU of IC323-Luci (circles), IC/Ed-P-Luci (squares), IC/Ed-L-Luci (triangles), and IC/Ed-PL-Luci (diamonds). After various intervals, the Renilla luciferase activities were measured. Data are means ± standard deviations for triplicate samples. RLU, relative light units. (B) Confluent monolayers of Vero/hSLAM cells in 6-cm culture plates were infected with 1.0 × 10⁴ PFU of IC323-Luci (light gray bars), IC/Ed-P-Luci (black bars), IC/Ed-L-Luci (dark gray bars), and IC/Ed-PL-Luci (white bars) and cultured in the presence of a fusion-blocking peptide. At 18 h p.i., mRNAs were purified from the cells, and the levels of N, P, M, F, H, and L mRNAs were determined by reverse transcription-quantitative PCR. Data are means ± standard deviations for triplicate samples. (C) Minigenome assays. The method was described in detail elsewhere (19). Monolayers of CHO/hSLAM cells cultured in Opti-MEM on 24-well plates were infected with vTF7-3 at a multiplicity of infection of 0.5 and then transfected with 0.2 μg of p18MGFLuc01-wt-Le, 0.2 μg of pCAG-T7-IC-N, 0.3 μg of a P-protein expression plasmid (pCAG-T7-IC-PΔC or -Ed-PΔC), and 0.2 μg of an L-protein expression plasmid (pGEMCR-IC-L or -Ed-L) using Lipofectamine 2000 (Invitrogen). At 6 h p.i., the culture media were replaced with RPMI 1640 medium supplemented with 7.5% fetal bovine serum. At 48 h p.i., the firefly luciferase activities were measured. (-), L-protein expression plasmid was omitted. Data are means ± standard deviations for triplicate samples.