Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

Abstract

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.

Lipid droplets and the host–pathogen dynamic: FATal attraction?

“…TBK1 phosphorylates IRF3, which migrates into the nucleus to promote expression of type I IFN. In parallel, the capacity of viperin to inhibit cholesterol biosynthesis by interaction with lanosterol synthase and squalene monooxygenase ( Grunkemeyer et al, 2021 ) could function synergistically to activate STING ( York et al, 2015 ) and to inhibit virus replication and virion release ( Wang et al, 2007 ). (3) In addition to viperin, numerous IFN-inducible proteins accumulate on LDs ( Bosch et al, 2020b ).…”

“…2 B ; Crosse et al, 2020 ). The capacity of viperin to inhibit cholesterol biosynthesis by interacting with key enzymes of the biosynthetic pathway that reside on LDs ( Grunkemeyer et al, 2021 ) could function synergistically to activate STING ( York et al, 2015 ) or to reduce the viral replication compartment formation ( Ilnytska et al, 2013 ; Strating et al, 2015 ). In conclusion, these studies indicate that viperin on LDs nucleates signaling platforms, produces antiviral molecules, and reduces cholesterol biosynthesis to enhance the synthesis of type I IFN and antiviral defenses.…”

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life

“…The viperin-associated restriction mechanisms employed against different viruses have been proposed to involve interactions between viperin and a wide range of viral and host proteins involved in diverse cellular functions, including metabolism, , signaling, iron–sulfur cluster formation, lipid raft perturbation, targeted protein degradation, and isoprenoid biosynthesis. ,, Two recent reviews have discussed these topics in depth. , Independent of its antiviral functions, viperin has also been demonstrated to play a physiological role in regulating thermogenesis and lipogenesis. , The breadth of these functions is not immediately suggestive of a common or shared mechanism for antiviral function. Furthermore, mechanistic details governing these putative interactions remain incomplete and are predominately based on indirect methods (e.g., yeast two-hybrid, colocalization, and immunoprecipitation), without direct validation by quantitative biochemical approaches, though several recent studies have begun to dissect these interactions in a quantitative manner. , …”

“…Furthermore, mechanistic details governing these putative interactions remain incomplete and are predominately based on indirect methods (e.g., yeast two-hybrid, colocalization, and immunoprecipitation), without direct validation by quantitative biochemical approaches, though several recent studies have begun to dissect these interactions in a quantitative manner. 26,27 As a member of the radical S-adenosyl-L-methionine (SAM) superfamily of metalloenzymes, 28 viperin employs a [4Fe-4S] (FeS) cluster cofactor that binds and supports homolytic cleavage of SAM, yielding methionine and a 5′-deoxyadenosyl radical (5′-dA•). 29,30 The only member of the radical SAM (RS) superfamily that does not catalyze this reaction is the cobalamin-dependent RS methyltransferase TsrM involved in the biosynthesis of thiostrepton.…”

Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs

“…This network of protein–protein interactions results in the enzyme exerting a wide array of antiviral properties beyond just the synthesis of ddhCTP. Cellular enzymes shown to interact with viperin include the mitochondrial trifunctional protein 10 , 11 , which is involved in fatty acid β-oxidation; squalene monooxygenase and lanosterol synthase 12 , 13 , which catalyze key steps in sterol biosynthesis; and interleukin receptor-associated kinase 1 (IRAK1) and the E3 ubiquitin ligase, TRAF6 14 – 16 , which are components of the TLR7/9 innate immune signaling pathway. Viperin also binds to a wide range of viral proteins from viruses such as hepatitis C, Dengue, tick-borne encephalitis and Zika viruses 2 , 8 , 9 .…”