FIG. 2.
Prematurely terminated mRNA was detected neither in the presence nor in the absence of VP30. (A) Northern blot analysis of EBOV-specific RNA-species. HeLa cells were infected with MVA-T7 and subsequently transfected with pT/NPEBO, pT/VP35EBO, pT/LEBO, pT/VP30EBO, and 3E-5EΔ250 as indicated. Forty-eight hours p.i., total RNA was isolated with TRIZOL reagent and purified either by using a biotinylated (biot.) primer coupled to immobilized streptavidin, which binds to the 5′ end of the minigenomic mRNA species (lanes 1 to 3), or by using oligo(dT) cellulose (lanes 5 to 7). Precipitated RNA was transferred onto nylon membranes and finally probed with the negative-sense digoxigenin-labeled riboprobe DIG-BS/CAT (27). The arrow indicates the position of the full-length mRNA species, which is 921 nt in length [without poly(A) tail]. (B) RNase protection assay. HeLa cells were infected and transfected as described above. Total RNA was isolated by using TRIZOL reagent. The radiolabeled negative-sense riboprobe comprised nt 1 to 270 of the EBOV leader (open bar) and 253 nt of upstream vector sequences (solid bar). Due to binding of the riboprobe to the 5′ end of the minigenomic mRNA (striped bar), a 214-nt segment of the probe was protected against RNase digestion. As a control, in vitro-transcribed positive-sense minigenome 3E-5E(+) was used for both experiments. Numbers indicate the length of the respective RNA marker bands. Probe −, riboprobe without RNase; Probe +, riboprobe with RNase.