Helical Ordering of Envelope Associated Proteins and Glycoproteins in Respiratory Syncytial Virus Filamentous Virions

Abstract

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.

Three-dimensional insights into human enveloped viruses in vitro and in situ

“…( E ) Central slice through a tomogram of a respiratory syncytial virus (RSV) filament (left). Crystal structure of the RSV matrix protein (PDB: 4V23) docked into a subtomogram average of the RSV matrix protein [ 71 ]; matrix protein (gold), subtomogram average (translucent grey) (right). ( F ) Composite structure showing the IAV M1 matrix protein (translucent grey and turquoise) (EMD-22384) forms the matrix layer over the RNP (not visible).…”

“…Recently there have been several high-resolution cryo-ET/STA studies reporting previously uncharacterized structures of matrix protein layers within purified virions, including HIV-1 [ 69 ], IAV [ 70 ], RSV [ 71 , 72 ], and Ebola virus [ 73 ]. Conley et al [ 71 ] and Sibert et al [ 72 ] used cryo-ET and STA to image RSV released from infected cells that were grown on EM grids.…”

“…Recently there have been several high-resolution cryo-ET/STA studies reporting previously uncharacterized structures of matrix protein layers within purified virions, including HIV-1 [ 69 ], IAV [ 70 ], RSV [ 71 , 72 ], and Ebola virus [ 73 ]. Conley et al [ 71 ] and Sibert et al [ 72 ] used cryo-ET and STA to image RSV released from infected cells that were grown on EM grids. Their results elegantly showed that the helical lattice-like sheet of M protein tightly associated to the inner leaflet of the membrane is reminiscent of the unit cell of the RSV matrix crystal structure and that M likely coordinates the helical ordering of pairs of viral glycoproteins [ 71 , 72 ] ( Figure 1E ).…”

“…Conley et al [ 71 ] and Sibert et al [ 72 ] used cryo-ET and STA to image RSV released from infected cells that were grown on EM grids. Their results elegantly showed that the helical lattice-like sheet of M protein tightly associated to the inner leaflet of the membrane is reminiscent of the unit cell of the RSV matrix crystal structure and that M likely coordinates the helical ordering of pairs of viral glycoproteins [ 71 , 72 ] ( Figure 1E ). Similar studies using cryo-ET and STA (∼8 Å) on IAV showed the matrix protein (M1) forms polymers in helical assemblies and provided insights into virus assembly and disassembly ( Figure 1F ) [ 70 ].…”

Interactions between the Nucleoprotein and the Phosphoprotein of Pneumoviruses: Structural Insight for Rational Design of Antivirals

“…The RNA is constrained and twisted by the N proteins, alternating rows of four and three stacked bases that are exposed and buried within the protein groove, respectively. Surprisingly, N-RNA rings, which were considered artifacts of production/purification, were recently found together with NCs in viral particles [101]. This raises the question of the potential role of these oligomers during the viral cycle.…”