Figure 3.

Monomeric E^rns cleaves dsRNA in denaturing conditions in vitro. Non-tagged C171 and R171 E^rns of the BVDV strain NADL were pre-incubated for 5 min in 20 mM sodium citrate buffer pH 6.0 that contained 7.2 M urea and 1 mM EDTA prior to the addition of 250 ng dsRNA of 300 bp in length of the NS3-region of the genome of BVDV strain Suwa. The reaction mixture was incubated for 60 min at 37 °C in a final volume of 8 μl. Medium (ctrl) and the supernatant of the HEK cells transfected with the empty vector (pCI-SN), used at equal volumes as that of the corresponding E^rns sample, served as a control for unspecific RNase activity. Finally, the RNA was separated on a 1% agarose gel containing ethidium bromide for 25 min at 100 V and visualized under UV light. One representative experiment out of three is shown.