Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo

Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro, in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.