The Molecular Basis of Aichi Virus 3A Protein Activation of Phosphatidylinositol 4 Kinase IIIβ, PI4KB, thrlough ACBD3

Phosphatidylinositol 4-kinase III beta (PI4KIII beta) is an essential enzyme in mediating membrane transport, and plays key roles in facilitating viral infection. Many pathogenic positive-sense single-stranded RNA viruses activate PI4KIII beta to generate phosphatidylinositol 4-phosphate (PI4P)-enriched organelles for viral replication. The molecular basis for PI4KIII beta activation during viral infection has remained largely unclear. We describe the biochemical reconstitution and characterization of the complex of PI4KIII beta with the Golgi protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3) and Aichi virus 3A protein on membranes. We find that 3A directly activates PI4KIII beta, and this activation is sensitized by ACBD3. The interfaces between PI4KIIIb-ACBD3 and ACBD3-3A were mapped with hydrogen-deuterium exchange mass spectrometry (HDX-MS). Determination of the crystal structure of the ACBD3 GOLD domain revealed a unique N terminus that mediates the interaction with 3A. Rationally designed complex-disrupting mutations in both ACBD3 and PI4KIII beta completely abrogated the sensitization of 3A activation by ACBD3.