Dengue virus protein NS3 activates hexokinase activity in SAT-390 hepatocytes to support virus replication Marianne FIGL, Clémence JACQUEMIN, Patrice ANDRE, Laure PERRIN-COCON, Vincent LOTTEAU, Olivier DIAZ International Center for Infectiology Research (CIRI), INSERM U1111, CNRS UMR5308, Université de Lyon, FRANCE 1 INTRODUCTION 4 RESULTS 5 CONCLUSIONS Result 2: DENV NS3 protein interacts with hexokinases Viruses are mandatory parasites that use metabolism machinery to Result 1: DENV efficiently replicates in HuH7 HK4+ HK2- cells A Protein Complementation Assay B Coimmunoprecipitation Even if NS3 is able to stimulates glycolysis in cells expressing replicate. Growing literature demonstrates that viruses manipulate A.A. DENV-NS3 versus human metabolism enzymes DENVB.B.-NS3 versus hexokinases A. HuH7 HuH7 HK4+ HK2- B. (a) (b) 60 Lysate Co-IP HK2 or HK4, we observe an higher DENV replication in HuH7 central carbon metabolism (CCM) and more specifically glycolysis for HuH7 HuH7 55 NS3-3xFlag - + - + HuH7 HK4+ HK2- HK4+ HK2- suggesting that HK4 positive cells are more susceptible their propagation [1]. However, the underlying mechanisms are not HuH7 HK4+ HK2- 50 HK1 α-Gluc 45 α-Flag to DENV replication. fully described. Our team has already demonstrated that hepatitis C 40 80 80 HK2 α-Gluc *** 35 NS5A protein interacts and activates hexokinases (HKs) to favor viral 70 70 α-Flag cells 30 Poster presented at: presented Poster Fluorescente light Fluorescente 60 cells 60 α-Gluc replication [2]. It was described that dengue infection (DENV) 25 HK3 We observed that HuH7 HK4+HK2- cells have a rewiring of their 50 50 α-Flag 20 increases glycolysis [3] and thus we wondered if control of 40 40 glycolytic pathway resulting in intracellular lipids accumulation (see 15 HK4 α-Gluc 30 hexokinase activity was shared by DENV, another Flavivirus. 30 10 α-Flag POSTER FRI-291). In these cells, HK-reaction product (Glucose-6- 20 * 20 *** 5 GLUD2 α-Gluc % DENV positive positive DENV % Visible light Visible 10 10 0 phosphate) is redirected toward pentose-phosphate pathway for % DENV positive positive DENV % α-Flag PC FH HK1 HK2 HK3 GCK TPI1 PFKL IDH1 IDH2 PFKP 0 FBP1 PGK1 PGK2 G6PC LDHC LDHB SDHC LDHA LDHD 0 SDHA GOT2 GOT1 ENO1 ENO2 ENO3 PDHB SDHD PFKM PKM2 PGM1 PGM2 OGDH IDH3B MDH1 MDH2 ECHS1 IDH3G GLUD2 PDHA1 GAPDH PGAM2 24h 48h 72h 72h SUCLA2 nucleotides synthesis which could favor viral replication. 2 AIM Time (hours post-infection) Time (hours post-infection) Moreover literature indicates that DENV interferes with cellular Potential interactions between CCM enzymes and DENV NS3 protein were analyzed Decipher molecular mechanisms that enable DENV to control C. To determine the impact of hexokinase switch on viral lipid metabolism for its replication [6]. The modification of by PCA. Normalized Luminescence Ratio (NLR) was highly positive for NS3/HK2 glycolysis. Mock replication, HuH7 or HuH7 HK4+HK2- cells were intracellular lipids induced by HK4 expression could, almost in part, + DENV and NS3/HK3 (Fig.A). infected at an MOI of 0.1. 72h post-infection, infected explain the enhanced viral replication. 2,5 Interactions between NS3 and the 4 isoenzymes (HK1, HK2, HK3 and HK4) were ** cells were observed (Fig. A) and percentage of infection ) confirmed by co-immunoprecipitation (Fig. B). HEK293T cells, were cotransfected determined by flow cytometry (Fig. B). Viral 3 METHODS 2 with plasmids of interest. Glutamate dehydrogenase 2 (GLUD2) protein was used as We observe that modulation of HK4 activity in these cells modulates propagation was higher in HuH7 HK4+ HK2-. Cellular Models infected 1,5 a negative control of interaction. DENV replication. In this context, direct interaction between NS3 DOI: 10.3252/pso.eu.ILC2019.2019 consumption DENV infection enhances glucose consumption (Fig. C). +HK4 gene mock and HK appears as a molecular mechanism for DENV to modulate Targeting of HK2 gene 1 of of NS3 interacts with the four hexokinase isoenzymes, by CRISPR/Cas9 transduction Interestingly, glycolysis increase was higher in HuH7 glycolysis for its replication. Glucose Glucose Fold the first limiting step of glycolysis. ( 0,5 HK4+ HK2- cells. 0 HK4 expression increases DENV replication. HuH7 HuH7 HK4+ DENV infection enhances glycolysis and Result 4: DENV replication depends on HK4 activity HK2- A. 6 ACKNOWLEDGEMENTS HK2 +HK4 depends on glucose concentration. 160 In normal cells, HK4 is negatively Financial support: Fondation pour la Recherche Medicale (FRM) (grant 140 ** regulated by GKRP. HK4-GKRP DEQ20160334893). Technical support: SFR Biosciences (UMS3444/CNRS, 120 Result 3: NS3 increases cellular glycolysis n.s. interaction can be stimulated by fructose- U8/Inserm, ENS de Lyon, UCBL) facilities,: AniRA-cytometry and –L3 100 0+ mM0 mM 6-phosphate (F6P) or inhibited by laboratory. of 4h (RLU) of HuH7 HuH7 HK4+ HK2- n.s. n.s. 80 5+ mM5 mM A. (a) n.s. (b) 2 2,5 n.s. 10+ 10 mM mM fructose-1-phosphate (F1P) [5]. We are grateful to Ralf Bartenschlager (University of Heidelberg, Germany) * n.s. Fold 60 1,5 2 Adjunction of fructose in cell culture for providing the DENV constructs. of protein) 40 of protein) 1,5 * HK2 : HK4 or Glucokinase (GCK) : 1 n.s. 1 20 increases intracellular F1P concentration. 0,5 glucose/µg - expressed in normal control) of (Fold 0,5 - expressed in hepatocellular glucose/µg (Fold of control) control) of (Fold Glucose consumption Glucose Interestingly, at 5mM glucose, DENV 0 1 2 0 consumption Glucose (µg of (µg 0 7 (µg of (µg HuH7 REFERENCES carcinoma (HuH7) hepatocytes and transduced in Mock NS3 full NS3 NS3 Mock NS3 full NS3 NS3 HuH7 HK4+HK2- replication is increased by fructose in a length protease helicase length protease helicase B. 1,2 HuH7 HK4+ HK2- cells B. (a) n.s. (b) dose-dependent manner (Fig. A), n.s. n.s. 2 2 n.s. ** ** [1] : Sanchez et al. 2014, ”Viral activation of cellular metabolism” - absent in HuH7 * * 1 whereas GKRP overexpression inhibits 1,5 1,5 of protein) of protein) ) replication (Fig. B). [2] : Ramière et al. 2014, ”Activity of hexokinase is increased by its interaction Preliminary observations 1 1 0,8 0,5 with hepatitis C virus protein NS5A” glucose/µg 0,5 Expression of a negative mutant for mock (Fold of control) control) of (Fold glucose/µg (Fold of control) control) of (Fold Lactate consumption Lactate Lactate consumption Lactate 0,6 ControlCtl 0 of 4h (RLU) (µg of (µg 0 Glucose-6-phosphate is mainly Glucose-6-phosphate actively of (µg HK4-GKRP interaction (D413A-GKRP) Mock NS3 full NS3 NS3 Mock NS3 full NS3 NS3 +pGKRP pGKRP [3] : Fontaine et al. 2015, ”Dengue virus induces and requires glycolysis for length protease helicase length protease helicase Fold 0,4 ( oxidized in glycolysis pathway fuels pentose phosphate pathway: Fold does not inhibit replication whereas optimal replication” C. (a) 1 2 3 4 (b) and generates excess of pyruvate increase of nucleotides and lipids HuH7 HuH7 HK4+ HK2- 1 2 3 4 GKRP expression does (Fig. B.a). 0,2 [4] : Cassonnet et al. 2011, “Benchmarking a luciferase complementation assay that is secreted. synthesis. 1 : +pControl F1P increases replication whereas F6P 1 : +pControl 2 : +pNS3 full lenght 0 for detecting protein complexes” Anti-3xflag Anti-3xflag 2 : +pNS3 full lenght inhibits it (Fig. B.b). 3 : +pNS3 protease 5mM 25mM 3 : +pNS3 protease Experiments 4 : +pNS3 helicase 4 : +pNS3 helicase C. (a) (b) [5] : Min Choi et al. 2013, ”Molecular basis for the role of glucokinase n.s. 120 Anti-actin Anti-actin 70 * regulatory protein as the allosteric switch for glucokinase” *** *** Infections were performed using full length GFP-DENV (genotype 2). 60 100 ContrôleControl Glucose consumption and lactate secretion in HuH7 (Fig. A.a et B.a) or HuH7 HK4+ Fructose+Fructose-1--1Phosphate-phosphate [6] : Heaton et al 2010, ”Dengue virus nonstructural protein 3 redistributes fatty Analysis of the effect of HK4 modulation on viral replication was 50 80 Fructose+ Fructose-6--Phosphate6-phosphate General hepatology Olivier Diaz HK2- (Fig. A.b et B.b) cells were determined after transfection for expression of ContrôleControl acid synthase to sites of viral replication and increases cellular fatty acid 40 performed using the subgenomic Renilla-DENV replicon. GKRP+pGKRP 60 NS3 or its domains. GKRP D413A synthesis” +pGKRP D413A of 4h (RLU) of 4h(RLU) of 30 40 20 Interactions between NS3 protein and CCM enzymes were revealed by Fold Full length NS3 but not its subdomains (helicase and protease) stimulates glucose Fold PCA (Protein Complementation Assay) realized in HEK293T cells [4] 10 20 consumption (Fig. A.a & b) and lactate secretion (Fig.B.a & b) in both cell types. and confirmed by co-immunoprecipitation (CoIP) 0 0 8 CONTACT Viral proteins expression after transfection was determined by western-blot (Fig. C). HuH7 HK4+ HK2- HuH7 HK4+ HK2- [email protected] SAT-390 NS3 stimulates glycolysis. HK4 activity modulation by GKRP controls DENV replication. ILC2019.