Dengue virus replication in hepatocytes depends on hexokinase isoform expressed

Le virus de la Dengue (DENV) est un arbovirus transmis par les moustiques du genre Aedes (Ae. aegypti et Ae. albopictus, communément appelé moustique tigre). Le DENV constitue un problème de santé publique majeure du fait de l’expansion du vecteur et pour lequel aucune solution thérapeutique ou vaccinale n’est disponible. Dans les formes sévères de la pathologie (fièvres hémorragiques), on observe une atteinte hépatique dont les causes sont multifactorielles et encore mal connues. Par ailleurs, stéatose hépatique et obésité constituent des facteurs aggravant la pathologie. Une littérature de plus en plus abondante montre que les virus sont capables de moduler le métabolisme glucidique et lipidique pour favoriser leur réplication. Il a également été observé que le DENV est capable de moduler le métabolique cellulaire pour favoriser sa réplication mais les mécanismes moléculaires sous-jacents sont peu caractérisés. Nous avons observé que l’expression de la protéine NS3 du DENV est capable d’induire une augmentation de la glycolyse dans des cellules d’hépatocarcinome HuH7. Nous avons également identifié l’interaction de NS3 avec les différentes hexokinases (HK), premières enzymes de la glycolyse. L’expression d’HK4, spécifique aux hépatocytes, dans des HuH7 restaure la lipogenèse et favorise la réplication virale. Nous avons également identifié l’interaction de NS3 avec GKRP, régulateur négatif d’HK4. La surexpression de GKRP ou la modulation de son activité sur HK4 par des facteurs métaboliques inhibe la réplication virale. L’ensemble des observations suggère une interférence entre la protéine NS3 et la régulation de l’activité HK4 par GKRP, favorisant ainsi l’infectionDengue virus (DENV) is an arbovirus which is transmitted by Aedes mosquitoes (Aedes aegypti and Aedes albopictus commonly known as tiger mosquitoes). This viral infection is today a major public health problem due to the spread of the vector and for which no therapeutic nor vaccine solution is available. In severe forms, which occur in 1 to 5% of cases (haemorrhagic fevers and life-threatening prognosis), liver damages are observed but their origins are still poorly understood and probably multifactorial. In addition, hepatic steatosis and obesity have been shown to be aggravating factors of the pathology. A growing literature shows that viruses are capable of modulating carbohydrate and lipid metabolisms to promote their replication. In case of DENV infection, it has also been observed that the virus is capable of modulating these cellular metabolic pathways to promote its replication, but the underlying molecular mechanisms are poorly characterized. We have observed that overexpression of DENV NS3 protein increases glycolysis in the hepatocarcinoma cells HuH7. We have also identified interaction of NS3 with the different cellular hexokinases (HK) that control the first step of glycolysis. The expression of HK4, normally and specifically expressed in hepatocytes, restores lipogenesis in HuH7 cells and promotes viral replication. We also identified NS3 as interacting with GKRP, the negative regulator of HK4. Overexpression of GKRP or modulation of its activity on HK4 by metabolic factors such as fructose-6-phosphate, inhibits viral replication. Overall, these observations suggest an interference between NS3 protein and the control of the first step of glycolysi

La réplication virale du virus de la dengue dans les hépatocytes dépend de l'isoforme d'hexokinase exprimée

Dengue virus (DENV) is an arbovirus which is transmitted by Aedes mosquitoes (Aedes aegypti and Aedes albopictus commonly known as tiger mosquitoes). This viral infection is today a major public health problem due to the spread of the vector and for which no therapeutic nor vaccine solution is available. In severe forms, which occur in 1 to 5% of cases (haemorrhagic fevers and life-threatening prognosis), liver damages are observed but their origins are still poorly understood and probably multifactorial. In addition, hepatic steatosis and obesity have been shown to be aggravating factors of the pathology. A growing literature shows that viruses are capable of modulating carbohydrate and lipid metabolisms to promote their replication. In case of DENV infection, it has also been observed that the virus is capable of modulating these cellular metabolic pathways to promote its replication, but the underlying molecular mechanisms are poorly characterized. We have observed that overexpression of DENV NS3 protein increases glycolysis in the hepatocarcinoma cells HuH7. We have also identified interaction of NS3 with the different cellular hexokinases (HK) that control the first step of glycolysis. The expression of HK4, normally and specifically expressed in hepatocytes, restores lipogenesis in HuH7 cells and promotes viral replication. We also identified NS3 as interacting with GKRP, the negative regulator of HK4. Overexpression of GKRP or modulation of its activity on HK4 by metabolic factors such as fructose-6-phosphate, inhibits viral replication. Overall, these observations suggest an interference between NS3 protein and the control of the first step of glycolysisLe virus de la Dengue (DENV) est un arbovirus transmis par les moustiques du genre Aedes (Ae. aegypti et Ae. albopictus, communément appelé moustique tigre). Le DENV constitue un problème de santé publique majeure du fait de l’expansion du vecteur et pour lequel aucune solution thérapeutique ou vaccinale n’est disponible. Dans les formes sévères de la pathologie (fièvres hémorragiques), on observe une atteinte hépatique dont les causes sont multifactorielles et encore mal connues. Par ailleurs, stéatose hépatique et obésité constituent des facteurs aggravant la pathologie. Une littérature de plus en plus abondante montre que les virus sont capables de moduler le métabolisme glucidique et lipidique pour favoriser leur réplication. Il a également été observé que le DENV est capable de moduler le métabolique cellulaire pour favoriser sa réplication mais les mécanismes moléculaires sous-jacents sont peu caractérisés. Nous avons observé que l’expression de la protéine NS3 du DENV est capable d’induire une augmentation de la glycolyse dans des cellules d’hépatocarcinome HuH7. Nous avons également identifié l’interaction de NS3 avec les différentes hexokinases (HK), premières enzymes de la glycolyse. L’expression d’HK4, spécifique aux hépatocytes, dans des HuH7 restaure la lipogenèse et favorise la réplication virale. Nous avons également identifié l’interaction de NS3 avec GKRP, régulateur négatif d’HK4. La surexpression de GKRP ou la modulation de son activité sur HK4 par des facteurs métaboliques inhibe la réplication virale. L’ensemble des observations suggère une interférence entre la protéine NS3 et la régulation de l’activité HK4 par GKRP, favorisant ainsi l’infectio

Domain 2 of Hepatitis C Virus Protein NS5A Activates Glucokinase and Induces Lipogenesis in Hepatocytes

International audienceHepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors

International audienceViruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.</div