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Ultrastructural characterization of a viral RNA and G-protein containing, membranous organelle formed in respiratory syncytial virus infected cells

Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants and the elderly. In common with most viruses that replicate in the host cell cytoplasm, RSV induces the formation of cytoplasmic compartments within infected cells to sequester replicative processes from host countermeasures. The best characterised organelle formed during RSV infection is the inclusion body – the primary site of viral RNA synthesis - thought to form as a membrane-less biomolecular condensate. Fluorescence microscopy of cellular compartments using probes directed at the structural proteins of RSV and the intergenic regions of the RSV genome have identified a second class of organelles termed assembly granules. Here we use correlative microscopy to identify assembly granules in the cytoplasm of frozen hydrated RSV infected cells for imaging using cryogenic soft X-ray tomography and cryogenic electron tomography. We show that these compartments are membrane bound, enclosing large numbers of vesicles, some of which contain RSV ribonucleoprotein complexes. Further we show that these organelles are frequently adjacent to mitochondria and surrounded by ER-like membranes. We also observe vesicles connected by junctions suggesting mixing of contents and a mechanism for the different viral proteins to come together within the assembly granule prior to budding. Collectively, our data provides novel insights into the RSV assembly process.