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 RNA-binding activity is important for the RNA helix-unwinding activity of helicase. We sought to assess whether the dsRNA-binding activity of VP35 can affect its RNA helix-unwinding activity. To this end, two VP35 mutants were constructed, including a deletion mutant that deletes the dsRNA-binding domain (named as interferon inhibitory domain, IID) of VP35 ( IID) (49) (50) (51) and the point mutant (RKR/AAA) that has its three critical dsRNA-binding sites (R305, K309 and R312) being mutated to alanine ( Figure 7A; Supplementary Figure S8 )(50). These mutant proteins were then expressed and purified as recombinant MBP-fusion proteins (Supplementary Figure S9) . Our results showed that both IID and RKR/AAA MBP-VP35 mutants completely lost their dsRNA-binding activities ( Figure 7B) . Moreover, to examine if VP35 can bind to EBOV-specific dsRNA, we ectopically expressed WT or RKR/AAA mutant Flag-VP35 in 293T cells together with EBOV 3 -UTR 1-200 dsRNA, followed by RNA-IP using anti-Flag antibody. Our data show that WT but not mutant Flag-VP35 could bind to EBOV 3 -UTR 1-200 dsRNA (Supplementary Figure S10) .