Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex

Abstract

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization.

Importance: hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.

FIG 1

The architecture of an hRSV viral particle. (A) A 7.5-nm central slice from a tomographic reconstruction of a filamentous hRSV viral particle. The boxed region highlights the area of the virus used in panel B. (B) Schematic of hRSV superimposed over the selected area of the hRSV viral particle from panel A. (C) Schematic representation of the architecture of an hRSV virus; the main structural components, the glycoproteins, the viral membrane, the matrix protein layer, the linker (M2-1), and RNP are indicated. Scale bars, 100 nm.

FIG 2

Basic morphological characterization of the three hRSV morphologies. Representative particles of the three morphology categories found in the RSV sample: spherical (A to C), asymmetric (D to F), and filamentous (G to I). Common structural features present in the 3D reconstructions of all hRSV morphologies include the surface glycoproteins, the viral membrane, the matrix protein layer, and RNP. Images are 7.5-nm central slices from tomographic reconstructions. Scale bars, 100 nm.

FIG 3

Subclassification of spherical hRSV viral particles. Top, models that represent the major structural features present in each group. Slices (4.5 nm) from tomographic reconstructions of spherical hRSV viral particles are presented for group 1 (A, D, and G), group 2 (B, E, and H), and group 3 (C, F, and I). Scale bars, 100 nm.

FIG 4

Linear profiles through hRSV particles. Slices (4.5 nm) from tomographic reconstructions of hRSV viral particles indicate the regions used to generate the linear profiles from the three hRSV morphologies (spherical [A], asymmetric [B], and filamentous [C]). The red dotted line marks regions with M, and the black dotted line marks regions without M. Linear profile graphs show the density peaks in the presence (red) or absence (black) of M in the viral particles. Peaks of the different structural components are marked in the density profile: surface glycoproteins (1), viral membrane (2), matrix (3), M2-1 (4), and RNP (5). Scale bars, 50 nm.

FIG 5

The RNP content of the hRSV viral particles. (A) Graph of the correlation between the traced RNP length and viral particle volume in the spherical, asymmetric, and filamentous virus morphologies. Based on previous studies, the average length of one full genome was calculated to be 1.48 μm. With this, the number of genomes per particle was determined. The left y axis notes the total RNP length in nm, the right y axis corresponds to the number of genome copies, and the x axis is the calculated volume of the viral particles. (B) Graph representing the frequency of the different genome numbers in the three RSV morphologies.

FIG 6

Effect of the matrix protein on hRSV particle morphology. (A) Graph of the amount of M coverage on the different particle morphologies. The number of particles included in the study and the P value of the t test are indicated. Mean and variance are marked in the table below. (B) Graph demonstrates the correlation between M coverage and surface area/volume ratio in the different particle types. (C) Relevant statistics for panel B are shown, displaying the number of particles in the study, R², and the P value for the linear regression model. Asterisks mark statistically significant correlation.

FIG 7

Spacing of M2-1 in hRSV viral particles collected using ZPC cryo-ET. (A) A 7.64-nm slice from a tomographic reconstruction of an hRSV viral particle used for measuring M2-1 spacing. Boxed region highlights the location of M2-1 densities in the slice. The inset is a magnified view of the boxed region; red dots mark the M2-1 densities. Scale bar, 100 nm. (B) Graph of the distances measured between M2-1 densities in three hRSV viral particles. The mean and standard deviation are marked.

FIG 8

M and M2-1 immunostaining and confocal fluorescence microscopy. (A) HEp2 cells infected with RSV A2 (MOI of 1) had RNA imaging probes targeting the RSV genomic RNA (red) delivered to them at 24 hpi and were fixed and immunostained for the viral F (blue) and M (green) proteins. Also included is a DAPI nuclear stain (gray). To the right, a merged image of an xy plane near the top of the cell is shown with orthogonal slices through the middle of the cell. Inset shows merged image of an xy plane near the bottom of the cell (to emphasize regularity of cell nuclei); boxed region shows magnified image of filamentous virions protruding from the cell membrane with the F and DAPI channels removed to emphasize the clarity of colocalization. Fluorescence intensity profile plot along arrow in the boxed region is also shown. (B) RSV-infected HEp-2 cells with RNA imaging probes (red) and immunostaining for viral F (blue) and M2-1 (green) proteins. (C) RSV-infected HEp-2 cells immunostained for viral F (blue), M (green), and M2-1 (red) proteins. Single optical plane, laser scanning confocal images shown. Scale bars, 10 μm.

FIG 9

dSTORM imaging of RSV filamentous virions. (A) Single RSV filamentous virion on glass, with RNA imaging probes targeting the RSV genomic RNA (green) delivered to it prior to isolation from cells and immunostained for viral M protein (red). A merged image of M and RNA and a side view are also shown. Magnified images of the boxed region in the side view are shown to the right. Arrow in the merge represents an example 100-nm cross section used to measure the axial FWHM. (B) Single RSV filamentous virion on glass with RNA imaging probes that was immunostained for the viral M2-1 protein. Views similar to that in Fig. 10A are shown. (C) Table containing axial FWHM of the point count for the viral protein and RNA in the 100-nm boxed region of the filaments shown in panels A and B. This measurement was repeated for 2 filaments for each protein. Scale bars are 1 μm for whole filaments.

FIG 10

Proximity ligation assay between F and M, F and M2-1, and M and M2-1. RSV virions on glass were assayed for interactions between F and M, F and M2-1, and M and M2-1 by PLA. The virions were also immunostained for pan-RSV for context. Note that PLA signal is an indication of two molecules in close proximity and may not colocalize with staining. From left to right, column 1 shows pan-RSV staining, column 2 shows PLA signal between F and M (A), F and M2-1 (B), and M and M2-1 (C), column 3 shows the merged image of columns 1 and 2 with pan-RSV signal in green and PLA signal in red, and column 4 shows the fluorescence intensity profile plot along the arrow. The first rows show filamentous virion morphologies, while second rows show spherical virion morphologies. Boxes indicate viral particles of interest; scale bars, 2.5 μm.

FIG 11

Proximity ligation assay between RNA and either M or M2-1. RSV virions labeled with FLAG-tagged MTRIPs on glass were assayed for interactions between FLAG and either M (A) or M2-1 (B) by PLA. The virions were also immunostained for pan-RSV for context. Note that PLA signal is an indication of two molecules in close proximity and may not colocalize with staining. From left to right, column 1 shows pan-RSV staining, column 2 shows RNA probes targeting the RSV genome, column 3 shows PLA between M and either FLAG-tagged or control MTRIPs (A, rows 1 and 2, respectively) and between M2-1 and either FLAG-tagged or control MTRIPs (B, rows 1 and 2, respectively), column 4 shows the merge of columns 1, 2, and 3 with pan-RSV signal in green, RNA signal in blue, and PLA signal in red, and column 5 shows the fluorescence intensity profile plot along the arrow. Scale bars, 2 μm.