The Study Of The NTPase And Helicase-like Activities Of Ebola Virus VP35

Ebola virus(EBOV)is a filamentous,enveloped,non-segmented,negative-sense RNA virus,belonging to the genus Ebolavirus in the family Filoviridae of the order Mononegavirales.EBOV is a BSL-4 pathogen and is notorious for its high pathogenicity and mortality rates(25-90%)in humans.The 2014-2016 Ebola outbreak in West Africa caused over 28,000 cases and 11,000 human deaths.Since May 2019,an on-going Ebola outbreak takes place in DR Congo,which has caused ~900 cases and over 600 deaths,and the situation is still deteriorating.Thus far,there is no approved drug or vaccine available to treat EBOV.Besides EBOV,non-segmented,negative-sense RNA viruses(NNSVs)are a huge class of viruses including 9 virus families and numerous important human and zoonotic pathogens,such as rabies virus,Nipah virus,Marburg virus,respiratory syncytial virus,human parainfluenza viruses,measles virus,mumps virus,etc.And these viruses share co mMon organizations of viral genomic RNAs and similar strategies of transcription and replication.For RNA viruses including NNSVs,viral RNAs contain multiple cis-acting elements that play critical roles in viral RNA replication,transcription,translation,and encapsidation.Similar with cellular RNAs,the functionalities of these highly structured RNA elements need proper folding and/or refolding,which are quite challenging and require the participation of a variety of viral or cellular RNA remodeling proteins,such as RNA helicases and RNA chaperones.These RNA remodeling proteins are thought to be indispensable for viral life cycles,making them key enzymatic requirements for efficient viral replication.However,to date no RNA helicase or chaperoning activities has linked with any filovirus(including EBOV)or NNSV proteins,and it is unclear as to whether the life cycle of filoviruses or NNSVs require the participation of these enzymes.This gap significantly hampers our understanding about this large group of important pathogens,as well as the development of antiviral drugs.Filoviral VP35 is an essential polymerase L cofactor that is analogous to the P proteins of other NNSVs.In addition,VP35 is a multifunctional protein that plays pivotal roles in filoviral replication and transcription,nucleocapsid assembly,as well as evasion of host antiviral defenses.In this study,we demonstrate that although containing no conserved NTPase/helicase motifs,EBOV VP35 has the NTP-binding,NTPase and helicase-like activities that can hydrolyze all types of NTPs and unwind RNA helices in an NTP-dependent manner.Moreover,we found that NTPase/helicase-like activities of VP35 can be inhibited by guanidine hydrochloride(GuHCl),a helicase inhibitor and FDA-approved small molecule and,more importantly,that it can inhibit the replication/transcription of an EBOV minigenome in cultured human cells.Together,these findings not only provide the first demonstration of the NTPase/helicase-like activities encoded by any member of NNSVs,but also highlight a key enzymatic activity that may be a viable antiviral target for EBOV.