Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus

Abstract

The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

Analysis and application of the SRBSDV P9-1 octamer crystal structure for the target of α-amino phosphonate derivatives

“…The eight subunits of the asymmetric unit were individually refined by simulated annealing, as indicated by the root mean square deviation (RMSD) between the individual subunits (0.01-0.02 Å). The secondary structure of each subunit in the refined atomic model was very similar to that described previously by Akita et al [28], with RMSDs of ~ 0.6 Å, ~1.6 Å and ~1.6 Å for the carbon-alpha backbone (not including the loop region). The residue of the monomer densities corresponding to fitted coordinates were supplied in Supplementary Figure 2.…”

“…The overall structure of SRBSDV P9-1 was similar to the RBSDV P9-1 octamer [28]. The SRBSDV P9-1 octamer was ~83.33 Å in height, ~95.14 Å in width ( Figure 1A) and ~119.51 Å in diameter ( Figure 1B).…”

“…The residue of the monomer densities corresponding to fitted coordinates were supplied in Supplementary Figure 2. The wellordered portions had similar secondary structure in all eight subunits in the octamer [28].…”

“…A typical octahedral-shaped crystal belongs to the space group C2221. The structure was solved by molecular replacement in Phaser [41] using the published RBSDV P9-1 monomer as a search model (PDB code 3VJJ) [28]. The SRBSDV P9-1 model was manually built in COOT [42], and computational refinement was conducted with the program REFMAC5 [43] in the CCP4 suite.…”

“…The μNS protein acts as a scaffold to recruit viral core surface proteins [25], while σNS binds preferentially to ss-RNA and ss-DNA [26,27]. In members of the genus fijivirus such as RBSDV, nonstructural protein RBSDV P9-1 is important for the functioning of the relative proteins in Vps during viral morphogenesis [28], RBSDV P9-1 self-associates into aggregate formations and preferentially binds ss-RNA molecules [29,30]. Given the high homology between SRBSDV P9-1 and RBSDV P9-1 [31], SRBSDV P9-1 could have a similar function, and may also be able to bind small molecules.…”