Spatial Uncoupling of Protein Synthesis from Viral Factories during Rabies Virus Infection

Replication and transcription of Mononegavirales (MNV) occur in cytoplasmic viral factories. In rabies virus (RABV) infected cells, these membrane-less, liquid-liquid phase-separated organelles are called Negri bodies (NBs). Their accumulation of viral mRNAs suggests that, in addition to supporting viral RNA synthesis, they may also serve as sites of viral protein translation. Using immunofluorescence and confocal microscopy, we demonstrate that ribosomal subunit proteins and translation initiation factors are excluded from NBs, including the ribosomal protein RPL40, a component of the large ribosomal subunit involved in the unconventional translation initiation mechanism of RABV. To investigate the spatiotemporal dynamics of protein synthesis during RABV infection, we performed pulse-labeling experiments with the methionine analogue homopropargylglycine (HPG). HPG labeling revealed a rapid accumulation of newly synthesized proteins within NBs, occurring independently of both microtubule and actin cytoskeletons. RABV infection also triggered the formation of stress granules (SGs), in which some ribosomal proteins and translation initiation factors are concentrated. Notably, a marked inhibition of protein synthesis was observed in SG-positive cells. Together, these findings indicate that in RABV infected cells translation occurs outside NBs, followed by the targeting of nascent viral proteins to these compartments.