DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Structural basis of viperin-induced changes in lipid synthesis upon virus infection – VipVir

Structural basis of Viperin-induced changes in lipid synthesis upon virus infection

Lipids are present at every step of the virus life cycle including entry, uncoating, transcription, translation, assemble and egress. They play a crucial role in virus replication and are thus interesting targets for the development of new antiviral strategies. The interferon stimulated genes are part of this strategy and in particular viperin, our protein of interest, seems to interfere with the lipid biosynthesis mechanism.

Viperin seems to act on the decrease of cellular cholesterol, leading to the inhibition of viral budding. We propose to discover the substrate and to decipher its mode of action on virus restriction.

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an interferon-stimulated gene that has antiviral activity against a variety of pathogenic enveloped viruses including cytomegalovirus, influenza A, dengue virus and HIV-1. Its precise mode of action is largely unknown, but accumulating evidence demonstrates its implication in lipid biosynthesis and membrane modulation affecting enveloped virus replication. Its main characteristic is to contain an iron-sulfur cluster in its central catalytic domain, namely S-Adenosylmethionine (SAM) radical domain. Radical SAM enzymes use the reduction of one electron of a Fe4S4 cluster to cleave SAM and produce methionine and a very reactive 5’-deoxyadenosyl (5’dA.) radical specie, which is in turn used to initiate radical-based chemistry on various substrates.<br />Our project focuses on three main objectives, the first one being to understand how the upregulation of viperin interferes with lipid biosynthesis process during influenza virus infection. We focus on the identification and the structural characterization of the viperin substrate as well as on the modified product of the same reaction. This will shed light on the mechanism of inhibition of cholesterol formation and thus viral budding.<br />The second objective was to study the interaction of viperin with the mitochondrial trifunctional protein (TFP), which is exploited by the Human CytoMegaloVirus (HCMV), which takes control of the lipid overproduction to enhance viral particle release through the inhibition of the TFP.<br />The third objective was to complement the structural work described in the 2 first objectives by functional studies and cell biology approaches to shed light on the function of viperin during influenza and cytomegalovirus infections.

We cloned and purify several constructs of human viperin as well as viperin from different homologues in aerobic conditions. These constructs contain or not the N-terminal amphipathic helix. Les full-length constructs needed all the addition of detergent, in particular DDM (N-dodecyl ?-D-maltoside) for their purification.
The reconstitution of the Fe4S4 cluster is performed with the addition of FeCl3 and Na2S in molar excess in an anaerobic environment, using the Jacomex glove boxes located in the metalloprotein lab headed by Yvain Nicolet at the Institut de Biologie Structurale, with whom we collaborate. All purification steps are, from this moment, performed in the absence of oxygen.
We performed crystallization tests with and without reconstitution of the cluster on some of our constructs thanks to the platforms at the IBS and at the PSB (Partnership for Structural Biology).
In order to identify the substrate of viperin reaction, we set up an enzymatic test to measure 5’dA radical formation by viperin in the presence of SAM and dithionite, a strong reducing agent. The reaction product is then purified and analyzed on HPLC coupled with mass spectrometry and fluorescence measurements with an excitation at 280nm and emission at 395nm, characteristics of 5’dA.
The inhibition test of FPPS, an enzyme from the mevalonate pathway, has been performed with the malachite green assay that quantify the pyrophosphates released during the reactions it catalyses. The pyrophosphates are converted into orthophosphates by pyrophosphatase and the colorimetric reaction with the malachite green is then monitored on a regular spectrophotometer at 620nm.

Our collaboration with the metalloprotein group at the IBS allowed us to optimize protocols on purification and reconstitution of different viperin’s constructs from human and from other homologous organisms. These protocols are now robust and we managed to characterize these different constructs with biochemical and biophysical techniques. In particular, we could show that the reconstituted falcon viperin has an increase in melting temperature of 10°C compared to the non-reconstituted one.
Crystallization tests for all these soluble constructs in reconstituted or non-reconstituted forms are actually in progress.
Several efforts have been raised on full-length viperin constructs from human and chinchilla, which contain the N-terminal amphipathic helix. Detergent is needed at all the steps of protein extraction and purification that are now mastered and crystallization tests are in progress.
Next, we tested different substrates on viperin’s activity and we discovered a potential substrate which is a product of the mevalonate pathway leading to cholesterol formation. This lipid precursor will be modified by the 5’dA-based radical chemistry reaction triggered by the reaction between viperin and SAM. This modified molecule will then block an enzyme from the mevalonate pathway, leading to the inhibition of cholesterol formation and thus virus budding. This major discovery will allow us to explain the viperin’s mechanism on enveloped virus budding.
The first tests of FPPS inhibition by this modified molecule did not show any inhibition of the FPPS and further tests on downstream enzymes of the mevalonate pathway will be tested.

Unexpectedly, we discovered one of the real substrates of viperin involved in the perturbation of the mevalonate enzymatic pathway leading to cholesterol production and thus in influenza virus restriction. Our next goals will be to identify the modification on the terpene substrate. We are actually collaborating with Amaury du Moulinet d’Hardemare from the Department of molecular chemistry at University Grenoble Alpes in order to identify this modified molecule by NMR, mass spectrometry and liquid phase chromatography techniques.
Once the molecule is identified, we will start crystallization of our viperin constructs (from human or other organisms) in complex with this molecule as a co-substrate with SAM in order to understand the chemical mechanism of viperin action.
Once the modified terpene molecule is known, we will also have to identify the enzyme of the mevalonate pathway that needs to be inhibited in order to monitor a decrease in cholesterol production. We will test several enzymes of the mevalonate pathway downstream FPPS by the above-described malachite green colorimetric assay and also test the binding of the modified terpene on the identified enzyme with Surface Plasmon Resonance (SPR) and MicroScale Thermophoresis (MST)
Finally, we will test the inhibitory effect of the product on HIV budding as we already have experience in the lab in expressing HIV-1 Gag in different cell lines. Gag expression is sufficient to produce virus-like particles (VLPs) that are released into the supernatant. The addition of the modified product of the viperin reaction is expected to be able to block the mevalonate pathway thus decreasing cholesterol formation and consequently inhibit VLP budding. VLP budding will be monitored by western blot by determining intracellular Gag versus released Gag. Reduction of VLP release will be further monitored by electron microscopy or cryo-electron tomography to detect budding defects.

N/A

Viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an interferon-stimulated gene that has antiviral activity against a variety of pathogenic enveloped viruses including cytomegalovirus, influenza A, dengue virus and HIV-1. Its precise mode of action is largely unknown, but accumulating evidence demonstrates its implication in lipid biosynthesis and membrane modulation affecting enveloped virus replication. Indeed, lipids are present at every steps of the virus life cycle including entry, uncoating, transcription, translation, assembly and egress. Thus, the modulation of membrane structure and composition is an attractive target for the immune defense system as well as for the development of new therapeutics.
In this proposal, I propose an integrated multi-level approach involving structural, molecular and in vivo cell biology in order to decipher the role of this new restriction factor and its implication in lipid homeostasis. My first objective is to understand how the upregulation of viperin interferes with the lipid biosynthesis process during influenza virus infection. Viperin expression leads to a blocking in viral budding, which has been attributed to the inhibition of the farnesyl diphosphate synthase (FPPS) by viperin, a cellular regulatory factor implicated in lipid homeostasis. This objective includes the functional characterization and the structure solving of viperin, a radical-S-adenosyl-L-methionine (SAM) enzyme, in complex with its [4Fe-4S] cluster, SAM cofactor and its FPPS partner, with the help of the anaerobic expression, purification and crystallization facilities available on our campus. This structural characterization will represent the first structure of a eukaryotic radical SAM enzyme and will help understanding the molecular details of the inhibition of FPPS by viperin. This will be complemented by structure-function studies described in objective 3, performed by my collaborators to better understand the effect of viperin upregulation on influenza budding. My second objective is to study the interaction of viperin with the mitochondrial trifunctional protein (TFP) which is exploited by the human cytomegalovirus, which takes control of the lipid overproduction to enhance viral particle release through the inhibition of the TFP. My third objective will be to complement the structural work described in objective 1 and 2 by functional studies and cell biology approaches to shed light on the function of viperin during influenza virus and cytomegalovirus infections.
Altogether, my work will provide new structural details on a eukaryotic SAM enzyme, its enzymatic reactions and its role in inhibiting key enzymes of the lipid homeostasis processes. The integration of the structural work with the cellular function of viperin during influenza virus and cytomegalovirus budding will provide crucial molecular details on its role in lipid biosynthesis perturbation during viral infection. This will contribute to the understanding of cellular factors acting during the innate immune defense to modulate virus infection.

Project coordination

Pauline Macheboeuf (Institut de Biologie Structurale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IBS Institut de Biologie Structurale

Help of the ANR 257,576 euros
Beginning and duration of the scientific project: - 42 Months

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