Structure and Double-Stranded RNA-Binding Activity of the Birnavirus Drosophila X Virus VP3 Protein

The birnavirus multifunctional protein VP3 plays an essential role in coordinating the virus life cycle and interacting with the capsid protein VP2, the RNA-dependent RNA polymerase VP1, and the double-stranded RNA (dsRNA) genome. Furthermore, the role of this protein in controlling host cell responses triggered by dsRNA and preventing gene silencing was recently demonstrated. Here, we report the X-ray structure and dsRNA-binding activity of the N-terminal domain of drosophila X virus (DXV) VP3. The domain folds into a bundle of three α-helices and arranges as a dimer, exposing to the surface a well-defined cluster of basic residues. Site-directed mutagenesis combined with electrophoretic mobility shift assays (EMSAs) and surface plasmon resonance (SPR) revealed that this cluster as well as a flexible and positively charged region linking the first and second globular domains of DXV VP3 are essential for dsRNA binding. Also, RNA silencing studies performed in insect cell cultures confirmed the crucial role of this VP3 domain for the silencing suppression activity of the protein.
This work was supported by the Spanish Ministry of Science and Innovation (BIO2017-83906-P and MDM-2014-0435 to N.V. and AGL2017-87464-C2-1-P to J.F.R.). D.S.F. acknowledges the contract from Banco Santander under agreement number 902030160036.