Designing Methods for Understanding Cell-Cell Communication in a Virus Microenvironment during Human Cytomegalovirus Infection

Abstract

For productive viral replication, both an infected cell and its surrounding environment are metabolically and defensively rewired. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus across the global population that is the leading infectious cause of birth defects and extensively remodels its surrounding microenvironment throughout infection. Cells proximal to an HCMV infection exhibit decreased immune signaling and increased viral protein abundance, priming them for a subsequent infection. In my thesis work, I sought to understand the mechanisms behind this remodeling of the viral microenvironment (VME) using a multidisciplinary approach including microscopy, proteomics and method development. Preliminary findings revealed that cell-cell contact dependent mechanisms are an important axis of intercellular communication deserving of more rigorous investigation. My project focuses on the role of mitochondria, tunneling nanotubes, and direct cell-cell contacts more broadly in altering the immune signaling pathways of cells proximal to a site of infection. I developed a cell line that labels mitochondria using HaloTag technology that allowed for the first known visualization of mitochondrial transfer during HCMV infection. Further exploring cell-cell communication within a VME, I leveraged fluorescence-activated cell sorting and proteomics to investigate the relative contributions of contact-mediated and contact-independent communication on bystander cells. Finally, I developed innovative strategies to visualize tunneling nanotubes, study the effects of cell density on immune signaling, and spatially pattern the VME. This work ultimately provides evidence that contact-dependent mechanisms of cell-cell communication during HCMV infection are crucial for modulating the immune response in neighboring cells.