Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 A resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 angstrom resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.

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.