Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infection is largely unknown. Here, we used barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 (CK1) in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and the 5′ trailer of genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host CK1-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted viroplasm formation and virus replication. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state for virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of the viral P protein and virus infection.