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

Abstract

f 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.

Structure and Function Analysis of Nucleocapsid Protein of Tomato Spotted Wilt Virus Interacting with RNA Using Homology Modeling

“…The protein was dialyzed in storage buffer (50 m m NaH 2 PO 4 , 300 m m NaCl, pH 8.0) and stored at −70 °C until use. To purify the free-tag TSWV N protein, we removed the N-terminal His 6 tag from the recombinant N protein, which was expressed from pET28a-N by a recombinant His 6 -tobacco etch virus protease as described by Wu et al ( 28 ).…”

Rice Reoviruses in Insect Vectors

“…The genesis and maturation of viroplasms of rice reoviruses are beginning to be understood using the development of vector cell lines (18,68,113,115). Initially, the nonstructural proteins of rice reoviruses such as RBSDV and SRBSDV P9-1, RDV Pns12, RGDV Pns9, and RRSV Pns10 can form octameric structures as a basic scaffold to initiate nascent viroplasm matrices (1,2,18,46,68,113,118,128); thus, these viral proteins are generally called viroplasm matrix proteins. Transgenic rice plants that can inhibit expression of viroplasm matrix protein genes show complete resistance against viral infection (90,96,97,98), suggesting that the blocking of viral replication site formation is a priority for controlling viral diseases.…”

Stability of local secondary structure determines selectivity of viral RNA chaperones

“…Other functionally analogous proteins encoded by different members of the Reoviridae family form octamers, similar to NSP2 (notably P9-1 and Pns9 proteins (67,69)). Despite their similar toroidal architecture, NSP2 presents a continuous, basic RNA binding groove on the surface, while P9-1 binds ssRNA within a positively charged inner pore.…”