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

RNA helix, DNA helix and RNA-DNA hybrids were prepared by annealing two complementary nucleic acid strands. One was labeled at the 5 end with hexachlorofluorescein (HEX)-labeled, and the other strand was unlabeled. HEX-labeled oligonucleotide strands were purchased from TaKaRa (Dalian, China). Unlabeled DNA strands were synthesized by Invitrogen, and unlabeled RNA strands were in vitro transcribed using T7 RNA polymerase (Promega, Madison, WI). The in vitro transcribed RNA strands were purified by Poly-Gel RNA Extraction Kit (Omega bio-tek, Guangzhou, China) according to the manufacturer's instructions. The two strands were mixed in a proper ratio, and annealed through heating and gradually cooling as previously described (21, 27) . The resulting duplexes were examined by 15% native-PAGE gels to make sure that all the single-stranded RNA or DNA was annealed in a ratio of 1:1. The standard (R*/R) by annealing a 42-nt HEX-labeled single -stranded RNA1 and a 54-nt nonlabeled single-stranded RNA2. The 3'-tailed and 5 -tailed RNA helixes by annealing a 42-nt HEX-labeled singlestranded RNA1 and a 48-nt non-labeled single-stranded RNA3 (3 -tailed) or RNA4 (5 -tailed). (D*/D) by annealing a 28-nt HEX-labeled single-stranded DNA1 and a 49nt non-labeled single stranded DNA2, (D/R*) by annealing a 42-nt HEX-labeled single-stranded RNA1 and a 30-nt non-labeled single-stranded DNA3, (R*/D) by annealing a 42-nt HEX-labeled single-stranded RNA1 and a 54-nt nonlabeled DNA4. All oligonucleotides used in this study are listed in Supplementary Table S2 .