The Study Of The Helicase And RNA Chaperone Activities Of Guaico Culex Virus NSP2

Guaico Culex virus(GCXV)is the firstly uncovered animal-infected multicomponent virus with segmented positive-sense genomic RNAs.GCXV belongs to the Jingmenvirus group,a diverse clade of segmented viruses that are related to the prototypically unsegmented Flavivirus.The discovery and construction of Jingmenvirus group is quite new,and current researches are focused on the discovery,identification and phylogenetic analysis of new viruses.However,little is known about the exact functions of the Jingmenvirus-encoded proteins.For most RNA viruses,including flaviviruses,the functionality of RNA molecules usually requires correct folding into proper high-order structures.Also,the proper structures of cis-acting RNA elements within the genomes of RNA viruses are essential for RNA replication,translation and encapsidation.However,because RNAs can automatically fold to a thermal stable-level state that is incorrectly structured and inactive,folding viral RNAs to the correct structure is quite challenging.In response,many viruses evolve to encode various RNA remodeling proteins,including RNA helicases and RNA chaperones,which can destabilize RNA duplexes' base-pairings,thereby promoting the proper correcting of RNA structures.RNA helicases contain adenosine triphosphatase(ATPase)activity and participate in ATP-dependent unwinding of structured RNAs,and they are classified into six superfamilies(SFs),designated SF1 to SF6 on the basis of conserved helicase motifs.Moreover,RNA chaperones are a heterogeneous group of proteins without conserved sequences or motifs,but they can destabilize RNA duplexes and assist in annealing more stable RNA structures independently of ATP.Although GCXV NSP2 had been previously found to be similar to flaviviral NS3 at the protein-level,its authentic activity is still unknown.In this study,we expressed and purified GCXV NSP2 as a fusion protein with maltose-binding protein(MBP)at its N-terminus via baculovirus expression system.After a series of in vitro experiments,our study showed that NSP2 encoded by GCXV has nucleoside triphosphatase(NTPase)activity,ds RNA-binding activity and ATP-dependent RNA helicase activity.Besides,we determine that the optimal biochemical reaction conditions for GCXV NSP2 and that it can unwind ds RNA in a bi-directional manner.Moreover,we demonstrate that NSP2 also contains an RNA-chaperone activity that is capable of remodeling structured RNAs and facilitating RNA strand annealing independently of ATP.Besides,the critical domains responsible for the RNA remolding activity of NSP2 are also investigated.RNA helicases and RNA chaperones are recognized as the two distinct classes of RNA remodeling proteins.Interestingly,we found that GCXV NSP2 functions as both an RNA helicase and an RNA chaperone.This study provides the first demonstration of the RNA helicase and chaperoning activities associated with NSP2 encoded by GCXV,the firstly discovered animal-infected multicomponent RNA virus.Our findings not only increase our understanding of GCXV and Jingmenvirus,but also shed lights on the important roles of RNA-remodeling protein in the viral life cycles of multicomponent RNA viruses.