Structural analysis of HCMV assembly and the associated cellular remodeling

Abstract

Human cytomegalovirus (HCMV) is a highly prevalent member of the Herpesviridae family that establishes lifelong latent infections and can cause severe disease in newborns and immunocompromised individuals. HCMV induces profound and persistent changes in host cell architecture and metabolism to facilitate production and dissemination of viral progeny while evading immune detection. The aim of my doctoral research was to investigate HCMV assembly and the associated organelle remodeling in human fibroblast cells using in-situ electron cryo-tomography (cryo-ET). A major focus of this work was the structural characterization of peroxisomes and their remodeling during HCMV infection. To overcome the challenge of identifying peroxisomes in human fibroblasts, a novel correlative light and electron microscopy (cryo-CLEM) workflow was developed, combining cryo fluorescence microscopy (cryo-FM) and focused ion beam (FIB) milling with cryo-ET. This integrated approach enabled targeted identification and three-dimensional peroxisomes characterization in a near-native state. Using PTS1-mCherry as a specific peroxisomal marker, this revealed that mammalian peroxisomes human fibroblasts lack the distinctive crystalline bodies commonly observed in plants and fungi, appearing as inconspicuous spherical vesicles. In addition, this study provides detailed insight into the morphology and dynamics of membrane-bound structures within the viral assembly compartment (vAC) governing tegumentation and secondary envelopment of viral capsids during HCMV replication. Overview tomograms, acquired at low magnification to visualize the cellular ultrastructure within a section of a whole cell, were used as complementary tools to provide comprehensive spatial context for the organization and interactions of viral and cellular components within the vAC. This revealed capsids at various stages of secondary envelopment and the presence of multiviral bodies (MViBs). Overall, this thesis contributes to our understanding of viral replication and organelle remodeling in human fibroblast cells during HCMV infection, by providing new methodologies for the identification and characterization of intracellular structures in a near-native state.