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

According to the conventional view, helicases contain NTPase activity, utilize the energy of ATP binding and hydrolysis to melt nucleic acid base-pairings, and are thought to participate in most ATP-dependent rearrangements of structured RNAs. Helicases are generally classified into six superfamilies (SFs), designated SF1 to SF6, on the basis of conserved helicase motifs. Interestingly, although EBOV VP35 does not contain any conventional helicase motifs (57, 58) , the NTPase/helicase-like activities of VP35 have most basic biochemical characteristics of canonical RNA helicases, including the NTP-dependency, the requirement of divalent metallic ion, and the directionality of helix unwinding, leading to the question whether the canonical conserved motifs are not necessary for a protein to possess the NTPase and RNA remodeling activities. Factually, Ectropis obliqua picorna-like virus (EoV) 2C protein contains the conserved motif A, which is commonly recognized as the core NTP binding and NTPase active site of SF3 helicases (21) , but the NTPase activity of EoV 2C is not dependent on this motif. In addition, the capsid protein VP5 of Helicoverpa armigera cypovirus-5 (HaCPV-5; genus Cypovirus, family Reoviridae) has the NTPase activity, which is also Mg 2+ -dependent and can hydrolyze all kinds of NTPs and dNTPs, but contains no conserved NTPase/helicase motifs (27, 47) . Thus, our current study, together with the previous studies, imply that some of the NTPase and RNA remodeling activities may not strictly rely on conserved motifs in linear sequences of amino acids, but probably determined by more sophisticated active sites formed in tertiary protein structures. In the current study, we have determined that the middle aa. 137-173 region of EBOV VP35 is critical for its NTPase activity. This middle region does not belong to the N-terminal oligomerization domain and the C-terminal IID, both of whose structures have been resolved (45, 50) . Interestingly, although the structure of the VP35 middle region has not been determined yet, the sequence alignment of VP35 proteins from multiple filoviruses, including Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Reston ebolavirus (RESTV), MARV, and Lloviu virus (LLOV) shows that this region is highly conserved within Filoviridae (Supplementary Figure S7A) . And our predicted secondary structure from PSI-blast based secondary structure prediction (PSIPRED) revealed that this region contains a ␤-strand and an ␣-helix (Supplementary Figure S7B ) (59, 60) . Besides, the motif scan by using the PROSITE profiles via the MyHits (https://myhits.isb-sib.ch/cgi-bin/ motif scan) suggested that the amino acid T149 of EBOV VP35 is a possible protein kinase C (PKC) phosphorylation site (61) . These analyses highlight the importance of this region in the structure and function of filoviral VP35s. Future studies should reveal whether the ␤-strand and ␣-helix as well as the putative PKC phosphorylation site are critical for the NTPase/helicase-like activity of VP35.