Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein.

RNA viruses encode multifunctional proteins to overcome limited genomic capacity and mediate diverse processes in viral replication and host cell modulation. The rabies virus P gene encodes full-length P1 protein and the truncated isoform, P3, which acquires phenotypes absent from P1, including interactions with cellular membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS). This gain-of-function suggests that isoform multifunctionality arises not only from discrete functions of protein modules/domains, but also from conformational regulation involving interactions of the globular C-terminal domain and N-terminal intrinsically disordered regions (IDRs). The precise mechanisms underlying gain-of-function, however, remain unresolved. Here, we compare the structure and function of P1 and P3, identifying isoform-specific long-range intra-protomer interactions between the IDRs and C-terminal domain that correlate with conformational states, LLPS behavior, and subcellular localization. Mutations in P3 that alter MLO interactions correspondingly modulate these interactions. P1 and P3 can interact with similar/overlapping sets of MLO-associated proteins and have similar LLPS capacity, but only P3 binds RNA, and this interaction correlates with gain-/loss-of-function mutations. Our findings reveal that conformational differences in isoforms regulate LLPS behavior and contribute to protein-RNA interactions, which controls access to host LLPS structures, uncovering a previously unrecognized strategy in P protein multifunctionality.