Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055.
J Biol Chem. 2020 Feb 28;295(9):2839-2849. doi: 10.1074/jbc.RA119.011526. Epub 2020 Jan 24.
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.
了解病毒逃避宿主细胞免疫防御的机制对于开发改进的抗病毒疗法很重要。出人意料的是，人类巨细胞病毒利用抗病毒的自由基 SAM 酶 viperin（病毒抑制蛋白，内质网相关，干扰素诱导）来增强病毒感染力。这个过程涉及到 viperin 向线粒体的易位，在那里它与三功能酶复合物的β亚基（HADHB）结合，该复合物催化β-酮酰基辅酶 A 酯的硫解，作为脂肪酸β-氧化的一部分。在这里，我们研究了这两种酶之间的相互作用如何改变它们的活性并影响细胞内的 ATP 水平。用纯化的酶进行的实验表明，viperin 抑制 HADHB 的硫醇酶活性，但出乎意料的是，HADHB 激活了 viperin，导致抗病毒核苷酸 3'-脱氧-3'，4'-二脱氢-CTP 的合成。从表达这些酶的转染的 HEK293T 细胞的裂解物中测量酶活性的实验结果与用纯化酶获得的结果一致。因此，viperin 定位于线粒体可降低硫醇酶活性，而 HADHB 的共表达显著增加了 viperin 的活性。此外，将 viperin 靶向线粒体也增加了 HADHB 逆行易位出线粒体并降解的速度，为 viperin 降低 HADHB 活性提供了另一种机制。将 viperin 靶向线粒体使细胞内 ATP 水平降低了 50%以上，这与该酶破坏脂肪酸代谢一致。这些结果为人类巨细胞病毒颠覆 viperin 的机制提供了生化见解；它们还为 viperin 最近在调节脂肪组织中的产热作用中发现的作用提供了生化依据。
