Structural analysis of virus assembly by cryo-electron tomography: Measles virus and respiratory syncytial virus

Abstract

Viruses, are such efficient nano-machines that they can pack all the genomic materials in a confined space essential for self-replication; and yet they are the smallest of living entities among all life forms. When studied carefully, viruses can parasitize in all living organisms. Animals, plants, bacteria, and fungi are all subject to viral infections. In this dissertation, I will be mainly focusing on investigating measles virus (MeV) and respiratory syncytial virus (RSV) by cryo-electron tomography (cryo-ET). MeV and RSV are single-stranded negative-sense RNA viruses. They are important human pathogens that can cause severe diseases; in some cases, they can lead to death. MeV and RSV are known for their pleomorphic nature. Because of the inherent heterogeneity, the structural studies of these two viruses have not been well explored until recent years. Whole-cell tomography of virus-infected cells is possible and provides a much more native environment to study virus assembly as well as the authentic architecture of viral particles. MeV remains a major human pathogen, with recurrent outbreaks impacting the pediatric population worldwide. To elucidate the principles governing paramyxovirus assembly and budding, we used cryo-ET to directly visualize MeV-infected human-derived cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) were characterized at stages of virus assembly and budding, and in released virus particles. A two-layered F-M lattice was revealed and the F-M lattice suggests that interactions between these proteins are present and may coordinate processes essential for MeV assembly. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly. RSV is the leading cause of lower respiratory tract disease in young children, immuno-compromised adults, and the elderly. In this study, we have used cryo-ET to study the morphology and assembly of RSV from infected human cells in its close-to-native state. Our results have demonstrated that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin sectioning TEM techniques. Taking advantage of the whole cell tomography technique, we have resolved different stages of RSV assembly. Collectively, our results will facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.