Respiratory Syncytial Virus P Has NTpase And Helicase-like Activities

Respiratory syncytial virus(RSV)is non-segmented,negative-sense RNA virus(Single-stranded,non-segmented,negative-sense RNA viruses,NNSV)in the family Pneumoviridae,and is a ubiquitous pathogen that can cause lower respiratory tract disease in the infants and high-risk adults.RNAs face significant challenges in efficiently folding into specific structures and switching between structural conformations.In addition,due to the limited diversity of RNA strands,unnatural base pairs are likely to be formed during the folding process,resulting in incorrect structures with no functional activity.Because these misfolded RNAs contain numbers of base pairs and double-stranded regions,they are difficult to be re-opened and folded into the correct conformation.To help misfolded RNAs to refold into the correct conformation,the host and virus encode RNA remodeling proteins.These proteins can help misfolded RNAs to open the hydrogen bonds between the paired bases and help them form the correct conformation to function correctly.According to the dependence of RNA remodeling proteins on NTP,they are divided into two categories: RNA helicase and RNA chaperone.For RNA viruses,virus-encoded RNA helicase plays critical roles during viral life cycles by facilitating structured RNA folding or refolding into the correct conformation.RSV is a pervasive pathogen that can cause severe respiratory diseases in infants,the elderly,and adults with weakened immune systems.The high incidence and prevalence of RSV infection,which results in everyone being infected with the virus at least once before the age of three,makes RSV a serious public health problem.The P protein of RSV is similar in function to the P/VP35 protein of other viruses in NNSVS.It is a multifunctional protein and can interact with polymerase L,nucleoprotein N,and matrix protein M2-1 to initiate viral transcription and replication.However,there is no report about whether RSV encodes helicases.In this thesis,we expressed RSV P recombinant protein and found that RSV P had NTPase activity and helicase-like activity,and could hydrolyze ATP,GTP,UTP.In addition,we further analyzed the biochemical characteristics and optimal conditions of RSV P.It was found that the NTPase and helicase-like activity of P must be produced by the presence of NTPs and metal divalent cations.Moreover,RSV P can unwind the 3'stranded end and 5' stranded ds RNA in the presence of ATP,and the unwinding activity of P to the 5' stranded ds RNA can be enhanced with the increase of ATP concentration.We further demonstrated the NTPase activity is required for the helixunwinding of RSV P.Together,our findings are the first demonstration of the NTPase and helicase activities of encoded by RSV,uncover novel functions of RSV P and highlight the significance of P in the life cycle of RSV.