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

The directionality of helix unwinding is a basic property of helicases. After finding that EBOV VP35 has the RNA helix-unwinding activity, we sought to assess its helix unwinding directionality. For this purpose, we used a classic assay by constructing three different RNA helix substrates that are 3 -tailed ( Figure 3A ), 5 -tailed ( Figure 3B ), and blunt ended ( Figure 3C ), respectively. The three helix substrates were then subjected to the RNA helix-unwinding assay by being reacted with purified MBP-VP35. Our results showed that MBP-VP35 efficiently unwound 5 -tailed RNA helix, but barely unwound 3 -tailed one ( Figure 3D ). Moreover, it could not unwind the blunt-ended RNA helix ( Figure 3E ), like other viral helicases such as EV71 2C (34) . To further determine the unwinding directionality of VP35, we evaluated its helix-unwinding activity in each direction in the presence of increasing concentrations of ATP. Our data showed that the increasing concentrations of ATP failed to promote the unwinding of 3 -tailed helix substrate ( Figure 3F ), but efficiently promoted the unwinding of 5tailed helix substrate in a dose-responsive manner ( Figure 3G and 3H). In conclusion, our data demonstrated that EBOV VP35 is able to unwind RNA helix in the directionality of 5 to 3 .