FIG. 4.
Characterization of RNA species synthesized by the artificial EBOV nucleocapsid complex. HeLa cells were infected with MVA-T7 and subsequently transfected with 500 ng of pT/NPEBO, 500 ng of pT/VP35EBO, and 2 μg of plasmid 3E-5E. One hundred nanograms of pT/VP30EBO and 1 μg of pT/LEBO were added to the transfection mixture as indicated. At 2 days p.i., cells were lysed. Lysates were either subjected to MCN digestion and subsequent RNA purification (A) or RNA was directly isolated from cell lysates and further purified by treatment with oligo(dT) cellulose (B). Purified RNA species were transferred onto nylon membranes and finally probed by using the negative-sense digoxigenin-labeled riboprobe DIG-BS/CAT (28). As a control, in vitro-transcribed positive-sense MBGV-specific minigenome 2.1-CAT was used (28) which is identical in size to 3M-5M. The arrows indicate the positions of positive-sense full-length RNA (A) or polyadenylated mRNA (B). NP, pT/NPEBO; 35, pT/VP35EBO; 30, pT/VP30EBO; L, pT/LEBO. (C) Influence of VP30 on CAT activity. HeLa cells were infected and transfected as described for Fig. 2. DNA transfection was performed with various amounts of pT/VP30EBO and fixed amounts of the other nucleocapsid protein genes (500 ng of pT/NPEBO, 500 ng of pT/VP35EBO, and 1 μg of pT/LEBO).