The Functional Study Of Norovirus Nonstructural Protein NS3

Norovirus,a non-enveloped,positive-stranded RNA virus,are highly contagious,infect people of all ages and cause vomiting,low-fever and acute gastroenteritis,even death for immunocompromised patients.Across the globe,approximately 18%acute gastroenteritis are caused by norovirus.Norovirus is the leading cause of food-borne disease and put a huge economic burden on countries around the world,especially developing countries.More severely,norovirus are threatening human health worldwide.The genome of norovirus consists of three open reading frames(ORF),ORF1 encodes six nonstructural protein,ORF2 and ORF3 encode two capsid proteins,VP1 and VP2 respectively.The six nonstructural proteins are crucial for viral life cycle,which are proved by previous studies.e.g.VPg functions as a protein primer to initiate the replication of viral genome,NS6 is a protease that processed the polyprotein into six mature proteins,and NS7 is a RNA-dependent RNA polymerase that synthesize the viral genome,However,there are still some proteins are not fully understood,such as NS3.For RNA viruses,their genome have to be strictly regulated to initiate their replication and translation.Futhermore,viral RNAs require correct folding into proper tertiary structure to play their roles on all kinds of biochemical process.Thus,RNA remodeling proteins-RNA helicase and/or RNA chaperone are required.According to amino acid sequence analysis,norovirus NS3 share conserved motifs with SF3 helicases.We hypothesize NS3 have putative helicase activity,therefore,we purified MBP-NS3 by baculoviurs expression system and perform a series of biochemical assays.We found norovirus NS3 have NTPase and helicase activity.It can unwind RNA duplex and RNA-DNA hybrids both from 5' to 3' and 3' to 5' in the presence of NTP and MgZ+.In addition,we found NS3 also have NTP-independent RNA chaperone activity,by which it can unwind two hairpin RNA and falicitate their annealing in vitro.Moreover,in vitro,NS3 can improve the efficiency of RdRP-dependent viral replication,which comfirmed its significant role on viral life cycle.Furthermore,we found guanidine hydrochloride(GuHCl)can inhibit the NTPase activity and helicase activity of NS 3 and inhibit the replication of noroviral replicon in human cells.NS3 can be a potential target for drugs and GuHCl is a candidate.Our study firstly comfirmed the helicase activity and RNA chaperone activity of norovirus NS3 and provide a potantial drugs for norovirus treatment.This study either proviedes thereotical foundation on norovirus research or pratical use for noroviral remedy.