Understanding the mechanisms by which viruses evade host cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus 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 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 synthesis of the antiviral nucleotide 3′-deoxy-3′,4′-didehydro-CTP. Measurements of enzyme activities in lysates prepared from transfected HEK293T cells expressing these enzymes mirrored the findings obtained with purified enzymes. Thus, localizing viperin to mitochondria decreased thiolase activity, and coexpression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB is retrotranslocated out of mitochondria and degraded, providing an additional mechanism by which viperin reduces HADHB activity. Targeting viperin to mitochondria decreased cellular ATP levels by more than 50%, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which human cytomegalovirus subverts viperin; they also provide a biochemical rationale for viperin's recently discovered role in regulating thermogenesis in adipose tissues.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 to 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

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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 bketoacyl-CoA esters as part of fatty acid β-oxidation. We have investigated how the interaction between these two enzymes alters their activities and their effect on cellular ATP levels. Studies with purified enzymes demonstrated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin to synthesize the antiviral nucleotide 3ʹ-deoxy-3′,4ʹdidehydro-CTP. Enzyme activities were also measured in lysates prepared from transfected HEK 293T cells transiently expressing these enzymes. Mirroring the studies on purified enzymes, localizing viperin to the mitochondria decreased thiolase activity whereas coexpression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB was retro-translocated out of mitochondria and degraded, providing an additional mechanism for reducing HADHB activity.Targeting viperin to the mitochondria decreased cellular ATP levels by over 50 %, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which HCMV subjugates viperin; they also provide a biochemical rational for viperin's recently discovered role in regulating thermogenesis in adipose tissues.