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

Since EBOV VP35 has dsRNA-binding, NTP-binding and NTPase activities, we speculated that VP35 may serve as a putative viral RNA remodelling protein. To examine if VP35 has RNA helix unwinding activity, we constructed the standard HEX-labeled RNA helix substrate with both 3 and 5 single-stranded tails by annealing a short 42-nt HEX-labeled RNA1 and a long non-labeled 54-nt RNA2 (as illustrated in Figure 2A) , which was commonly used to determine the helix-unwinding activity previously (34) . The helix-unwinding assay was performed by incubating the RNA helix substrate with MBP-VP35 in a standard unwinding reaction mixture, followed by the separation of the substrate strands via gel electrophoresis. Our data showed that the HEX-labeled RNA strand was efficiently released from the RNA helix substrate in the presence of MBP-VP35 ( Figure 2B, lane 4) , whereas the same helix substrate was not separated when the negative control (MBP alone) was present ( Figure 2B, lane 3) . Of note, the unwinding reactions were performed in the reaction mixture containing ATP and MgCl 2 ; and the boiled helix substrate or the addition of MBP-fusion EV71 2C, a well-characterized viral RNA helicase, was used as positive control for helix unwinding. And the helix-unwinding activity of VP35 is almost as robust as that of EV71 2C ( Figure 2C ).