Nucleolar targeting of lyssavirus P-protein is isoform- and phylogroup-specific.

The nucleolus is a multifunctional hub and a common target of viral proteins, yet its role in infections by cytoplasmically replicating RNA viruses remains poorly defined. In rabies virus (RABV), the phosphoprotein (P-protein) isoform P3 localizes to nucleoli and inhibits rRNA biogenesis, whereas P1 lacks nucleolar targeting, even when forced into the nucleus. Here, we show that nucleolar targeting is an isoform- and phylogroup-specific property of lyssavirus P-proteins. Isoforms P3-P5 accumulate in nucleoli, whereas P1 and P2 are excluded. Comparative analyses revealed that P3 nucleolar targeting is conserved in phylogroup I but absent in phylogroup II lyssaviruses. Co-immunoprecipitation assays identified conserved interactions with nucleolin and nucleophosmin (NPM1) but divergent binding to Treacle and nucleolar and coiled-body phosphoprotein 1 (NOLC1). These findings define nucleolar targeting as a gain-of-function of truncated P isoforms, demonstrate its conservation across phylogroup I lyssaviruses and suggest broader engagement with membraneless compartments, highlighting potential therapeutic vulnerabilities.