On the Role of VP3-PI3P Interaction in Birnavirus Endosomal Membrane Targeting

Birnaviruses are a group of double-stranded RNA (dsRNA) viruses infecting birds, fish and insects. Early endosomes (EE) constitute the platform for viral replication. Here, we study the mechanism of birnaviral targeting of EE membranes. Using the Infectious Bursal Disease Virus (IBDV) as a model, we validate that the viral protein 3 (VP3) binds to phosphatidylinositol-3-phosphate (PI3P) present in EE membranes. We identify the domain of VP3 involved in PI3P-binding, named P2 and localized in the core of VP3, and establish the critical role of the arginine at position 200 (R 200 ), conserved among all known birnaviruses. Mutating R 200 abolishes viral replication. Moreover, we propose a two-stage modular mechanism for VP3 association with EE. Firstly, the carboxy-terminal region of VP3 adsorbs on the membrane, and then the VP3 core reinforces the membrane engagement by specifically binding PI3P through its P2 domain, additionally promoting PI3P accumulation. Significance Statement Birnaviruses are a family of viruses unique among the group of dsRNA viruses. Here, we show that VP3 seizes the endosomes by anchoring to the PI3P lipids in the luminal side of the EE membrane, in order to organize the viral replication factory. We propose a two-stage modular mechanism for VP3 association with PI3P-enriched EE. The significance of this work resides in the biochemical and biophysical insights on the mechanism of association of VP3 with EE and its role in the viral life cycle, which represents a substantial contribution toward understanding the replication strategy of these “ non-canonical ” viruses.