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 a-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. IMPORTANCE The birnavirus moonlighting protein VP3 plays crucial roles in interacting with the dsRNA genome segments to form stable ribonucleoprotein complexes and controlling host cell immune responses, presumably by binding to and shielding the dsRNA from recognition by the host silencing machinery. The structural, biophysical, and functional data presented in this work identified the N-terminal domain of VP3 as being responsible for the dsRNA-binding and silencing suppression activities of the protein in drosophila X virus.

Automated summary

VP3 is a multifunctional protein in birnaviruses, essential for coordinating the virus life cycle and interacting with various viral components. The N-terminal domain of VP3 in drosophila X virus is responsible for binding to dsRNA and suppressing host gene silencing responses. This study provides structural and functional insights into the crucial roles of VP3 in birnaviruses.