A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation. Although linked to several viral processes, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.