HCMV infection depends on EGLN1-mediated mitochondrial activation to increase dNTP pools for viral DNA replication.

Human cytomegalovirus (HCMV) is a leading cause of congenital infection and morbidity in immunosuppressed populations. Like all viruses, HCMV is an obligate intracellular parasite that extensively remodels host cell metabolism to support its replication, yet the precise underlying mechanisms and the potentially associated metabolic vulnerabilities remain poorly understood. Using a metabolism-focused screening platform, we identify EGLN prolyl hydroxylase activity as critical for HCMV infection. Our studies reveal that HCMV infection depends on EGLN1, which accumulates in mitochondria during infection. Inhibition of EGLN1 expression blocks HCMV-mediated mitochondrial activation, which in turn prevents the production of the deoxynucleoside triphosphate (dNTP) precursors necessary for dNTP pool expansion and viral DNA replication. Further, pharmacological EGLN inhibition attenuates viral infection in a humanized mouse model. Collectively, these data establish EGLN1 as a critical determinant of mitochondrial metabolic remodeling and virally-induced dNTP generation during HCMV infection, highlighting EGLN1 as a promising antiviral therapeutic target.