Proximity-Driven Cell Cycle Reprogramming in Uninfected Cells within the Human Cytomegalovirus Microenvironment

Abstract

Human cytomegalovirus (HCMV) is a widespread β-herpesvirus that can cause serious disease and lifelong deleterious effects in people worldwide, especially in immunocompromised individuals and newborns. While extensive work has characterised how HCMV remodels cellular processes within infected cells, less is understood about how infection shapes the behaviour of surrounding uninfected cells in the microenvironment. Increasing evidence suggests that HCMV infection creates a spatially structured virus microenvironment (VME) in which cells neighbouring infected cells exhibit distinct phenotypes compared with cells that are more distal. In particular, prior work from our lab has shown that uninfected cells neighbouring an infection display marked cell cycle dysregulation, including increased S-phase entry and mitotic accumulation. This work also demonstrates that neighbouring cells are more susceptible to secondary and co-infection from multiple virus types than distal cells. These observations raise the possibility that HCMV actively reprogrammes the cell cycle of neighbouring cells to create conditions that favour viral spread. In this study, I investigated how these proximity-dependent changes in cell cycle behaviour vary across the HCMV microenvironment and the consequences of these dysregulations on genome stability. Using FUCCI-based live-cell imaging, I tracked cell cycle progression in uninfected cells within the HCMV microenvironment observing these cell cycle dysregulations in real time. To assess whether these changes were associated with genomic instability, I quantified γH2AX as a marker of double-stranded DNA breaks. Proximal uninfected cells exhibited elevated γH2AX signal compared to more distal populations, supporting the idea that premature S phase entry may lead to replication stress and induce double-stranded breaks. Together, these findings demonstrate that HCMV infection induces proximity-dependent changes in uninfected cells directly affecting central cellular processes such as the cell cycle. This work highlights the importance of considering the VME as an active and dynamic system and provides insight into how local cellular reprogramming may facilitate viral spread.