Targeting viperin to the mitochondrion inhibits the thiolase activity of the trifunctional enzyme complex

Abstract

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.

Viperin: An ancient radical SAM enzyme finds its place in modern cellular metabolism and innate immunity

“…This interaction was further investigated in vivo by directly targeting viperin to the mitochondrion by appending a mitochondrial leader sequence to the enzyme. Localizing viperin to the mitochondria decreased the thiolase activity of cell extracts, consistent with the in vitro experiments, and consequently caused cellular ATP levels to fall significantly (50). These effects may partly be attributed to viperin binding HADHB and inhibiting its activity, but targeting viperin to the mitochondrion also lowered HADHB protein levels, most likely by increasing the rate at which HADHB was retro-translocated out of mitochondria and degraded by the proteasome.…”

“…protein secretion and thermogenesis), which are unrelated to the innate immune response, it seems reasonable to ask whether there may be additional functions for ddhCTP. One recently suggested role is that ddhCTP, synthesized when viperin is translocated to the mitochondria, may alter the transcription of mitochondrial genes by acting as a chain terminator of mitochondrial mRNA (50,77).…”

“…Studies using purified enzymes in vitro showed that viperin directly binds to the b-subunit of MTP (HADHB) and inhibits the thiolase activity, which is performed by this subunit of the multienzyme complex (50). This interaction was further investigated in vivo by directly targeting viperin to the mitochondrion by appending a mitochondrial leader sequence to the enzyme.…”

“…These effects may partly be attributed to viperin binding HADHB and inhibiting its activity, but targeting viperin to the mitochondrion also lowered HADHB protein levels, most likely by increasing the rate at which HADHB was retro-translocated out of mitochondria and degraded by the proteasome. These functions appear to depend on the enzyme possessing a [4Fe-4S] cluster, as a viperin mutant lacking the [4Fe-4S] cluster still bound HADHB but failed to stimulate HADHB degradation, decrease thiolase activity, or depress cellular ATP levels (50).…”

“…Surprisingly, although viperin inhibits HADHB, HADHB was found to activate viperin toward the synthesis of ddhCTP severalfold (50). It is unclear why viperin should be activated to synthesize ddhCTP in mitochondria because this is not a site of viral replication.…”