Ebola virus VP35 has novel NTPase and helicase-like activities

Shu, Ting; Gan, Tianyu; Bai, Peng; Wang, Xiaotong; Qian, Qi; Zhou, Hui; Cheng, Qi; Qiu, Yang; Yin, Lei; Zhong, Jin; Zhou, Xi

Gel mobility shift assay was performed in 50 mM HEPES-KOH (pH 8.0), 100 mM NaCl, 2 mM MgCl 2 , 1 mM tRNA, 2 mM DTT, 20 U RNasin, in a total volume of 10 l reaction with the indicated amount of proteins and 0.1 pmol dsRNA or ssRNA. The dsRNA and ssRNA were labeled with DIG-UTP (Roche) by in vitro transcription and derived from 200-nt EGFP. Reactions were incubated for 30 min at 25 • C. The reactions were terminated by the addition of 2.5 l 5× sample buffer [20 mM Tris-HCl (pH 8.0), 30% glycerol and 0.1% bromophenol blue]. The nucleic acid-protein complexes were separated by electrophoresis on 1.5% agarose gels and transferred to Hybond-A nylon membrane (GE Healthcare). After that, the membrane was subjected to cross-linking with 120 • C and was incubated with anti-DIG-alkaline phosphatase antibody (Roche), followed by incubating with CDP-STAR (Roche) for 15 min at 37 • C. The signals are then detected by X-ray film (Fujifilm, Tokyo, Japan).