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

For most RNA viruses including NNSVs, viral genomic, antigenomic and messenger RNAs contain multiple cisacting elements that play critical roles in viral RNA replication, transcription, translation, and packaging during viral life cycles (14, 15) . Similar with cellular RNAs, these highly structured RNA elements need to be properly folded to be functional. However, the correct folding of RNA molecules is challenging, since RNAs could be kinetically trapped in misfolded states that are relatively stable in thermodynamics (16, 17) . To facilitate the correct folding of RNAs, a variety of RNA remodelling proteins, such as RNA helicases and RNA chaperones, are encoded by cells and viruses. These proteins function to destabilize RNA-RNA or RNA-DNA base-pairings to aid RNAs' proper folding or refolding (18) (19) (20) . In addition, they are believed to participate in the unwinding of viral doublestranded RNA (dsRNA) intermediates produced during replication and/or transcription, thereby facilitating the recycling of viral RNA templates for more efficient RNA synthesis. Thus far, a wide range of positive-sense RNA viruses and dsRNA viruses, including picornavirus (21) , norovirus (22) , flavivirus (23, 24) , alphavirus (25) , coronavirus (26) and reovirus (27) , have been found to encode their own RNA helicases and/or RNA chaperones (28, 29) . However, it is unknown whether NNSVs including filoviruses encode any protein with RNA remodelling activity or the replication/transcription of NNSVs requires the participation of any viral RNA remodelling activity. This apparent discrepancy hampers our understanding of this large group of important pathogenic viruses. Filoviral VP35 is an essential polymerase L cofactor that is analogous to the P proteins of other NNSVs (5). In addition, VP35 is a modular multifunctional protein that contains dsRNA-binding and oligomerization domains, and plays pivotal roles in filoviral replication and transcription, nucleocapsid assembly, and evasion of host antiviral defenses (7, (30) (31) (32) (33) (34) . Here, we show that EBOV VP35 has an unexpected nucleoside triphosphatase (NTPase) activity, which can hydrolyze all kinds of ribonucleotide triphosphates (NTPs). Strikingly, EBOV VP35 can function like a helicase to unwind RNA helices from 5 to 3 in an NTP-dependent manner. Moreover, we have found that guanidine hydrochloride (GuHCl), a well-known helicase inhibitor (22, 34, 35) and a U.S. FDA-approved small compound to treat the symptoms of muscle weakness and fatigability associated with Eaton-Lambert syndrome (https://www.accessdata.fda.gov/scripts/cder/daf/index. cfm?event=overview.process&ApplNo=001546) (36, 37) can inhibit the NTPase and helicase-like activities of VP35 in a dose-dependent manner. More importantly, GuHCl has also been found to exhibit inhibitory effect on the replication/transcription of a stable EBOV minigenome replicon in cultured human cells, highlighting the func-tional significance of VP35 s NTPase and helicase-like activities in EBOV life cycle, and implying that targeting the helicase-like activity of VP35 can be a novel strategy to develop antivirals against EBOV.