FIG. 3.

The NP gene start region is involved in formation of a stable secondary structure. In vitro-transcribed RNAs of positive-sense minigenomes 3E-5E(+) (left-hand side) and 3E-5E-NheI(+) (right-hand side), respectively, were subjected to chemical modification assay with the drugs DMS (A and C specific) and CMCT (U and G specific). In order to visualize minor RNase contaminations, samples were also incubated in the respective buffer without addition of DMS or CMCT, respectively. Modified RNA was purified and analyzed by the primer extension method. In parallel, the corresponding DNA sequence was determined by sequencing with the same radiolabeled primer. Samples were separated on a denaturing 8% polyacrylamide gel, and the gel was processed for autoradiography. Note that the cDNA fragments synthesized from modified RNA are displaced by 1 nt relative to the sequencing lanes. The sequence of the NP gene start region is shown below the autoradiographs. The conserved NP transcription start signal is underlined; modified nucleotides are marked by asterisks. Mutated nucleotides in the sequence of minigenome 3E-5E-NheI(+) are highlighted by gray boxes. The predicted and experimentally determined secondary structures of minigenome 3E-5E(+) are shown schematically on the right-hand side. Nucleotides belonging to the conserved transcription start signal are boxed. Nucleotide numbers refer to the genome sequence of EBOV Zaire.