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


OBJECTIVE
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.
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
Gabriella
G
Kiss
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
Jens M
JM
Holl
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
Grant M
GM
Williams
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
Eric
E
Alonas
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
Daryll
D
Vanover
Vutara, Inc., Salt Lake City, Utah, USA.
Aaron W
AW
Lifland
Vutara, Inc., Salt Lake City, Utah, USA.
Manasa
M
Gudheti
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
Ricardo C
RC
Guerrero-Ferreira
Research Technologies Branch, Microscopy Unit, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, Montana, USA.
Vinod
V
Nair
Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia, USA.
Hong
H
Yi
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Barney S
BS
Graham
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
Philip J
PJ
Santangelo
Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia, USA erwrigh@emory.edu.
Elizabeth R
ER
Wright
D020285:Cryoelectron Microscopy / Q000379:methods; D006801:Humans; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D018357:Respiratory Syncytial Virus Infections / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000502:physiology / Q000648:ultrastructure*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000378:metabolism
D012367:RNA, Viral; D012261:Ribonucleoproteins; D014763:Viral Matrix Proteins