Viroplasm protein P9-1 of Rice black-streaked dwarf virus preferentially binds to single-stranded RNA in its octamer form, and the central interior structure formed by this octamer constitutes the major RNA binding site.
The P9-1 protein of Rice black-streaked dwarf virus (RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed for in vitro binding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.
Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), Department of Plant Pathology, Nanjing Agricultural University, Nanjing, People's Republic of China.
Jianyan
J
Wu
Jia
J
Li
Xiang
X
Mao
Weiwu
W
Wang
Zhaobang
Z
Cheng
Yijun
Y
Zhou
Xueping
X
Zhou
Xiaorong
X
Tao
D020816:Amino Acid Motifs; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D012087:Reoviridae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins