Multi-modal imaging of respiratory syncytial virus genome-rich bodies

Respiratory syncytial virus (RSV) is an enveloped virus with a negative sense, single-stranded RNA genome. RSV is responsible for upper respiratory tract infections such as rhinitis and croup mainly in babies and young children, but it can also infect the elderly and the immunocompromised. If RSV infection progresses into the lower respiratory tract then more severe diseases such as bronchiolitis and pneumonia can occur, which can lead to death. There is no licensed RSV vaccine and the only licensed antivirals are monoclonal antibodies, such as palivizumab, which are for prophylactic treatment only. During the RSV lifecycle, intra-cytoplasmic compartments named inclusion bodies (IBs) form. It is thought that RSV carries out transcription and replication within IBs. IBs contain viral proteins, viral RNA and some cellular host proteins. They have also been described to form through phase-phase separations and exist as membraneless structures. Here we employed a multi-modal correlative imaging approach to explore RSV cytoplasmic compartments during RSV infection, aiming to better understand RSV replication. Our use of RSV genome-specific fluorescent probes discovered a new RSV-induced cytoplasmic organelle: genome-rich bodies, GRBs. GRBs appear to be distinct from IBs, containing much of the viral genome within the cell as evidenced through confocal laser scanning microscopy. Using soft X-ray cryotomography we found that GRBs are granular, membranous cytoplasmic structures. Cryo-focussed ion beam milling followed by electron cryotomography revealed that GRBs are enclosed by an outer lipid bilayer and contain many internal membrane bound compartments, some of which have multiple membrane layers. Within the internal compartments are ring-like and tubular structures which we hypothesise are RSV nucleocapsids - viral genomes and anti-genomes coated by the RSV nucleoprotein. Morphologically identical structures were also seen within RSV filaments budding out of infected cells. The application of multiple imaging modalities has enabled the discovery of GRBs on different resolution scales, generating a detailed description of GRB structure and morphology. Further investigation to explore mechanistic aspects of these structures may inform the development of future RSV interventions.