Definition of mechanisms of viral protein multifunctionality reveals key roles of ‘conformer diversity’ and RNA interaction in forming host interfaces

Multifunctionality of viral genes is critical for processes in replication and modulation of infected cells. P gene of rabies virus generates the full-length protein, P1, and the truncated isoform, P3, which gains unique phenotypes lacking in P1, including interactions with multiple cellular membrane-less organelles (MLOs, liquid-liquid phase-separated (LLPS) structures), important to immune evasion. The gain-of-function by P3 proposes that multifunctionality of P isoforms is not merely due to their complement of independent modules, but is regulated by complex interactions of globular and intrinsically disordered regions (IDRs). However, the molecular basis of gain-of-function is unknown. Here we report biophysical and cellular analyses of P1 and P3, identifying a network of intra-protomer interactions involving the globular C-terminal domain and N-terminal IDRs, which differ between the isoforms. Mutagenesis of P3 identified substitutions causing gain- and loss-of-function for MLO interactions, associated with altered interactions of N- and C-terminal regions. Despite reduced MLO association of P1 and P3-loss-of-function mutants compared with wild-type P3, they retain capacity for LLPS in vitro, suggesting that specific inter-molecular interactions enable MLO targeting. P3 and P1 interact similarly with multiple MLO-associated proteins, but RNA binding is only observed for P3, and is enhanced or diminished by gain- and loss-of-function mutations, respectively. These data indicate that differences in interfaces formed between distant regions in P protein isoforms regulate protein-RNA interactions as a principal mechanism in the acquisition of unique functions/MLO interactions by P3, identifying a novel strategy in viral protein multifunctionality.