Timestamping Integrative Approach to Understand Secondary Envelopment of Human Cytomegalovirus

The mechanisms facilitating the assembly of Human cytomegalovirus (HCMV) in the cytoplasm of infected cells, a complex process termed ‘secondary envelopment’, are poorly understood. Our goal is to identify in-situ the identity, position, and interactions of all the essential proteins involved in this critical stage of the viral ‘lifecycle’. Despite decades of research, it has been difficult to dissect the complexity of secondary envelopment, as bulk assays only show ensemble averages of populations of viral particles. To study these intermediates that are formed when cytoplasmic capsids acquire tegument proteins and their envelope membrane, we will develop a novel approach that separates these intermediates in time and space. We will provide their spatio-temporal models by integrating complementary cutting-edge techniques and expertise within this collaboration, including flow-virometry, correlative (fluorescence and electron cryo) microscopy, crosslinking and ion-mobility mass spectrometry-based proteomics, and computational modelling. Specifically, we aim to: -Identify key players in tegument assembly on capsids/membranes. -Elucidate the order and spatial organisation of tegument assembly. -Validate the interactions in vivo and analyse capsid tegumentation in vitro. -Integrate the information into a spatiotemporal model. This will significantly improve our understanding of herpesvirus assembly in general, a crucial step towards identifying new therapeutic targets.