Donut-shaped decamers and dodecamers from Mal de Río Cuarto virus P9-1 reveal the structural versatility of a major viroplasm protein (Comunicación Oral)

GABRIELA LLAUGER; ROBERTO MELERO; DEMIÁN MONTI; GABRIELA SYCZ; CRISTIÁN HUCK-IRIART; MARÍA L. CERUTTI; SEBASTIÁN KLINKE; ROCÍO ARRANZ; FERNANDO A. GOLDBAUM; YANN G. STERCKX; JOSÉ M. CARAZO; MARIANA DEL VAS; LISANDRO H. OTERO

Banff

Simposio; 14th International dsRNA Virus Symposium; 2022

University of Alberta

The fijivirus Mal de Río Cuarto virus (MRCV) causes a devastating maize disease. The non-structural protein P9-1 is the major component of the viroplasm where virus replication and packaging occur. The study of viroplasm structure, function, and dynamics of plant reoviruses lags far behind those of animal reoviruses. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures in vivo and for the protein multimerization in vitro. Employing an integrative structural approach involving X-ray crystallography, Cryo-EM, and SAXS, along with biophysical studies in solution, we showed that P9-1 dimerizes through hydrophobic interactions and hydrogen bonds. While the C-arm is dispensable for dimer assembly, it is essential for the formation of higher-order oligomers where dimers interact with each other through their C-arms giving rise to a population of donut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops facing the inner pore, where a potential RNA binding site and a PEST sequence (conditional proteasome-mediated degradation signal) are located. Reconstruction by molecular dynamics simulations of the loops that were not experimentally defined, revealed that the pore is more occluded in the decamer than in the dodecamer, suggesting that each assembly has different properties. Overall, our work presents direct evidence of two previously unidentified quaternary arrangements of a major viroplasm protein, paves the way for functional studies and establishes the structural basis for the development of antiviral strategies against MRCV.