The Structure And Biochemical Functions Of Viroplasm Protein P9-1 Of RBSDV And SRBSDV Bind To Single-stranded RNA

Rice black-streaked dwarf virus (RBSDV), Southern Rice black-streaked dwarf virus(SRBSDV) are both the members of the genus Fijivirus within the family Reoviridae. The two viruses each contains 10 double-stranded RNA genome segments,ORF1 of segment 9 encodes a nonstructural protein P9-1 with a molecular mass of 40 kDa. RBSDV P9-1 and SRBSDV P9-1 are viromplasm proteins, Viroplasm, which is believed to be the specific site of viral replication and assembly occur, contained the proteins, viral RNAs,outer-caspid proteins, core particles and mature particles. Viroplasm proteins form cytoplasmic inclusion structures by itself, once their expression are inhibited, the viroplasms will not be formed any more. Then the replication and assembly of virus are broken. Based on this function, viroplasm proteins are the essential part of the infection life cycle of virus, and they are the good target for disease control. One of the major biological functions of viroplasm proteins is that they can recruit and bind viral RNA transcripts for the synthesis of genomic dsRNA. However, little is know about the nature or biological functions in the viroplasm of the two P9-1 proteins, and we analyzed the structure and biological functions of RBSDV P9-1 and SRBSDV P9-1 for binding to ssRNA by biochemical experiments in vitro,1 The structure and biochemical functions of RBSDV P9-1 binds to ssRNAThe RBSDV P9-1 was expressed and purified with nickel-nitrilotriacetic acid resin.Purrified P9-1 protein was analyzed for its structure and biochemical functions of binding to nucleic acids in vitro. Agarose gel electrophoretic mobility shift assay (EMSA) and competition and specificity assays of RBSDV P9-1 suggested that the protein binds preferentially to single-stranded rather than double-stranded nuleic acids, but P9-1 protein does not have a preference for binding to RBSDV sequences. More than one unit of RBSDV P9-1 can bind each molecule of RNA which formed a range of different-size intermediate complexes. Both discontinuous blue native PAGE and chromatography analysis demonstrated RBSDV P9-1 forms dimers, tetramers, and octamers, and the dimer is the vast majority of the protein. Protein-RNA copmplexes were analyzed by gel electrophoretic mobility shift assay in discontinuous blue native PAGE, we found RBSDV P9-1 protein preferentially binds to ssRNA as an octamer, we also propose an octamer binding model. According to the P9-1 crystal structure, deletion of RBSDV P9-1 carboxy-terminal arm, the mutation protein no longer forms an octamer and binds to ssRNA with lower affinity. By using the online RNA binding residue prediction software BindN, we found that amino acid residues 25 to 44 and 319 to 347 were the major RNA binding domain. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and octamer formation, and when we mapped aa 25 to 44 onto crystal structure of P9-1, we found that these amino acids correspond to the central interior structure in the P9-1 octamer. Our findings provide a novel insights into the structure and funcntion of the viroplasm protein from oher viruses in the family of Reoviridae.2 The structure and biochemical functions of SRBSDV P9-1 binds to ssRNAThe amino acid sequence identity of RBSDV P9-1 and SRBSDV P9-1 was high(77%),and the previous research showed SRBSDV owns a new kind of inclusion body, therefore our study focus on the the structure and biological functions of SRBSDV viroplasm protein P9-1. We obtained amounts of SRBSDV His-tagged P9-1 protein for biochemical characterization by bacterial expression. The protein which purified with Ni-NTA resin was separated on 4-16% discontinuous blue native PAGE. SRBSDV P9-1 was capable of forming dimers, tetramers, heaxamers, octamers by self-assembly, and the difference is that with RBSDV P9-1, the octamer is the vast majority of SRBSDV P9-1. Agarose gel electrophoretic mobility shift assay and discontinuous blue native PAGE showed that SRBSDV P9-1 has a ssRNA binding activity, and the octamer is the form of RNA binding.The carboxyl terminus is important for protein structure, deletion of the last 23 aa of C-terminal, the SRBSDV P9-1 no longer forms octamers, and the RNA binding affinity is lower than wild-type protein. Alanine substitution analysis showed that the four positively charged amino acids, R26, R39, K43 and K44 among amino terminus are the RNA binding sites, the mutation protein almost losts the RNA binding activity, and forms a very small octamers. Based on the alignment of amino acids between the SRBSDV P9-1 and RBSDV P9-1,a major difference region is amino acids residues 131 to 160. The 131-160aa of RBSDV P9-1 affected the octamer formation and the RNA binding activity of RBSDV P9-1. It suggested that the match between amino acids of the protein is important for octamer formation and protein conformation, and the conformation is required for binds to RNA with high affinity. We proposed that the RNA binding activity depends on protein conformation and RNA binding sites, an exact match of conformation and binding sites leads to the maximized affinity.