An NMR Study Of The Capsid Protein Of Japanese Encephalitis Virus

Japanese encephalitis virus(JEV)is an mosquito-bone belonging to the Flavivirus genus of flaviviridae family.It is the main cause of viral encephalitis in many countries in Asia,and there are no specific antiviral drugs for Japanese encephalitis.JEV consists of three structural proteins and seven non-structural proteins.Among them,capsid protein C is the core protein of JEV particles,it is a structural element required for the assembly of the virus nucleocapsid and has the classic function of protecting the viral genome.In 2019,the structure of the capsid protein of Japanese encephalitis virus has been determined by X-ray crystallography,but no disordered N-terminal was observed in the crystal structure.According to reports,a single point mutation in the N-terminal of the capsid(N15K)has been reported to enlarge the virion and stabilize the vector,which may affect its assembly,but the structural basis of its N-termial remains unknown.Therefore,this paper uses NMR to study the solution structure of the JEVC protein.Firstly,the secondary structure of the JEVC protein and its oligomeric state in solution were measured by far-ultraviolet circular dichroism spectroscopy and analytical ultracentrifugation,which showed that there is α helix in the solution of the JEVC protein and its oligomeric state is a dimer.After that the JEVC protein was labeled with 2H/13C/15N and conventional triple resonance spectra were collected for backbone assignment.On the basis of backbone assignment,the secondary structure of the JEVC protein was predicted by TALOS+.The results show that the secondary structure of the JEVC protein in solution is similar to the crystal structure,and there are 4 alpha helices.At the same time,the longitudinal(T1)and transverse(T2)relaxation times of backbone 15N of the JEVC protein was measured,which further vefified the oligomeric state of the JEVC protein in solution.This research laid a foundation for the later use of NMR to obtain the dynamics information of the JEVC protein,and also laid the basis for the further mechanistical studies of virus packing and its interaction with host proteins.Studies have shown that the JEVC protein can interact with LC3-I,which is a non-lipidated form of microtubule-associated protein 1 light chain 3(MAP1LC3,LC3).The interaction between the JEVC protein and LC3-I was investigated by NMR based on backbone assignment.It shows that the interaction between the JEVC protein and LC3-I in solution is weak.This research laid the foundation for the subsequent study of its interaction in a membrane-regulated or a cell-like enviroment,and also provided a new idea for JEV infection.