Molecular Mechanism Of Nucleocapsid Protein Of Tomato Spotted Wilt Virus Interacting With Viral Genomic RNA

Tomato spotted wilt virus(TSWV)is the type species of the Tospovirus,the only genus in the family of Bunyaviridae that infects plants.TSWV causes serious diseases in numerous agronomic and ornamental crops worldwide and ranks among one of the most devastating plant viruses.Nucleocapsid(N)of TSWV is the main structural protein that assembles the genomic RNAs into ribonucleoprotein complexes(RNPs).The RNPs are central to the viral cycle of TSWV and other Bunyaviruses because only the RNPs,but not the naked RNAs,serve as the template for viral genome replication and gene transcription.Using yeast two-hybrid assay,TSWV N protein monomers was shown to interact with each other in a head-to-tail fashion.The TSWV N protein was also shown to bind single-stranded RNA irrespective of sequence.However,the molecular details of TSWV N-N multimerization and the interaction of TSWV N oligomers with genomic RNA remain to be elucidated.Although both halves of N were shown to be involved in RNA binding,the RNA binding sites on the N protein also remain to be determined.The TSWV genomic RNA is always associated with N protein;however,little is known about the protective function of the N-RNA complexes.The links between N self-interaction and characterization of N binding genomic RNA remain to be elucidated.The crystal structure of protein is an effective method to investigate biological function.There are more than 20 species of viruses in the Tospovirus genus,but none of the protein structures for these viruses have been determined.Fortunately,the crystal structure of N proteins from human and animal bunyaviruses has been determined in recent years.Based on these crystal structure of N proteins,we built the homology model of TSWV N protein.From the 3D structure we found some valuable basis to design experiments to evaluate the structure and analyze the function of the N protein.Research focused on the following three aspects:1.TSWV N forms a range of higher ordered oligomers.In this study,we developed blue native gel analysis,homology modeling,and other approaches to investigate the TSWV N-N oligomerization.TSWV N forms a range of oligomeric with different molecular size,such as monomer,dimer,trimer,tetramer,and pentamer.Deletional analysis of TSWV N protein reveals C-terminal 26 amino acids are responsible for protein interaction.F242 and F246 located in C-terminal 26 amino acids are critical for N self-interaction.N oligomers are highly dynamic.The monomer formed a dimer,whereas the dimer dissociated into the monomer.Nor did the trimer and tetramer maintain a stable form.The trimer dissociated to the dimer and monomer,and the tetramer was also able to convert to lower-order oligomers.2.The RNA binding sites and protection function of TSWV NIn this study,we investigate the interaction of TSWV N protein with genomic RNA by using homology modeling and other methods.Analysis of the RNA binding behavior of N protein using two different approaches revealed that no specific oligomer binds to RNA preferentially,instead each type of N oligomer is able to associate with RNA.Competition and specificity assays for N protein suggesting that N protein binds nonspecifically to RNA and displays no preference for the viral termini sequence.To better characterize the N protein in its interaction with genomic RNA,we constructed homology models of TSWV N and N-RNA complexes.Based on these homology models,we mapped the RNA binding sites onto its predicted surface cleft of TSWV N.Moreover,by N-RNA homology modeling we found that the RNA component is deeply embedded in the protein cleft of N;consistently,RNase A treatment assay revealed that TSWV N-RNA complexes are relatively resistant to digestion by RNase.3.The links between N binding RNA and N self-interaction.Discontinuous blue native PAGE analysis of oligomerization state PEI-treated N protein and RNA binding defective mutant N protein,we found that higher ordered oligomeric formation of the N protein does not depend on the binding of RNA.EMSA and RNase A treatment assay shows that the ability of N binding and protecting genomic RNA was affected by its oligomer states.