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bioRxiv · 10.1101/824425

Targeting Viperin to the Mitochondrion Inhibits the Thiolase Activity of the Trifunctional Enzyme Complex

Dumbrepatil, A. B.·Zegalia, K. A.·Sajja, K.·Kennedy, R. T.·Marsh, E. N. G.
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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 (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 {beta}-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes the thiolysis of {beta}-ketoacyl-CoA esters as part of fatty acid {beta}-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 3-deoxy-3,4-didehydro-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 co-expression 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 viperins recently discovered role in regulating thermogenesis in adipose tissues.

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Dumbrepatil, A. B., Zegalia, K. A., Sajja, K., Kennedy, R. T., Marsh, E. N. G.. 2019-10-30. Targeting Viperin to the Mitochondrion Inhibits the Thiolase Activity of the Trifunctional Enzyme Complex. https://doi.org/10.1101/824425

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