Abstract Birnaviruses form a distinct class of double-stranded RNA (dsRNA) viruses characterized by the absence of a transcription-competent inner core particle. The early endosomes (EE) of cells infected with the infectious bursal disease virus (IBDV) - a prototypical birnavirus and an important avian pathogen - constitute a platform for viral replication. Here, we study the mechanism of birnaviral hijacking of EE membranes for this process. We demonstrate that the viral protein 3 (VP3) specifically binds to phosphatidylinositol-3-phosphate (PI3P) present in EE membranes. We identify the domain of VP3 involved in PI3P-binding and its role in viral replication. Finally, our molecular simulations results unveil a two-stage modular mechanism for VP3 association with EE. Firstly, the carboxy-terminal region of VP3 adsorbs to the membrane via non-specific electrostatic interactions. Then, in the second stage, the VP3 core seals the membrane engagement by specifically binding PI3P through its P2 domain, additionally promoting PI3P accumulation. Significance Statement Birnaviruses are different from the rest of the dsRNA viruses. They contain an RNA-dependent RNA polymerase (RdRp) with a unique motif arrangement in the palm subdomain, their viral particles lack the inner protein layer protecting the dsRNA, and the viral capsid protein VP2 presents a jelly-roll topology characteristic of +sRNA viruses. Here, we provide evidence that birnaviruses replicate in association with cellular membranes. Since the remodeling of the host’s membranes is a characteristic shared by all +sRNA viruses, our results highlight parallels in the replication strategy of these “ non-canonical ” dsRNA viruses and +sRNA viruses.

Birnaviral Hijacking of Endosomal Membranes