Understanding the mechanisms by which viruses evade host-cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus (HCMV) co-opts the antiviral radical SAM enzyme, viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), to enhance viral infectivity. This process involves translocation of viperin to the mitochondrion where it binds the β-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes the thiolysis of β-ketoacyl-CoA esters as part of fatty acid β-oxidation. Here, we investigated how the interaction between these two enzymes alters their activities and affects cellular ATP levels. Experiments with purified enzymes indicated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin leading to the synthesis of the antiviral nucleotide 3'-deoxy-3',4'-didehydro-CTP. Measurements of enzyme activities in lysates prepared from transfected HEK 293T cells expressing these enzymes mirrored the findings obtained with purified enzymes. Thus, localizing viperin to the mitochondria decreased thiolase activity and co-expression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB is retro-translocated out of mitochondria and degraded, providing an additional mechanism by which viperin reduces HADHB activity. Targeting viperin to the mitochondria decreased cellular ATP levels by > 50 %, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which HCMV subverts viperin; they also provide a biochemical rationale for viperin's recently discovered role in regulating thermogenesis in adipose tissues.,Published under license by The American Society for Biochemistry and Molecular Biology, Inc.