cGAS activation during human cytomegalovirus infection is driven by exogenous DNA

Type I interferon (IFN) induction is a central component of the innate immune response to viral infection, and the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) has been identified as a key mediator of IFN production during human cytomegalovirus (HCMV) infection. However, how cGAS detects HCMV remains unresolved, as the viral genome is encapsidated and trafficked directly to the nucleus, limiting cytoplasmic exposure. Here, we show that IFN induction during HCMV infection of primary fibroblast cells is predominantly driven by cGAS recognition of exogenous DNA present in standard laboratory virus preparations rather than the encapsidated viral genome. DNase treatment of AD169 and low-passage TB40/E-GFP viral stocks substantially reduced total DNA content without affecting infectivity, yet markedly abrogated IFN induction, IFN-stimulated gene expression and IRF3 nuclear translocation. Immunofluorescence analysis further revealed cytoplasmic accumulation of DNA in cells infected with untreated virus stocks, which was absent following DNase treatment. Together, these findings demonstrate that contaminating DNA in viral preparations is sufficient to activate cGAS and drive IFN responses during HCMV infection in vitro, highlighting a critical confounding factor in studies of innate immune sensing. Author SummaryHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes lifelong infection and can cause serious disease in immunocompromised individuals and newborns. When cells detect viral infection, they produce type I interferons (IFNs), antiviral molecules that help limit virus spread. Previous studies have suggested that HCMV is sensed by a cellular DNA sensor called cGAS, which detects viral DNA in the cytoplasm and triggers IFN production. However, how cGAS gains access to the HCMV genome has remained unclear, because the viral DNA is enclosed within a protective capsid and transported directly to the nucleus during infection. In this study, we show that most IFN production observed during HCMV infection of fibroblast cells in vitro is driven not by sensing of the viral genome itself, but by contaminating DNA present in standard laboratory virus preparations. Treating virus stocks with DNase to remove this exogenous DNA abolished IFN induction without affecting viral infectivity. These findings highlight the importance of controlling for exogenous nucleic acids when interpreting how host cells detect viral infection.