Metabolic Reprogramming Of Host Cells In Response To JEV Infection And A Preliminary Intervention Via Targeting Metabolism

BackgroundJapanese encephalitis(JE),also named epidemic encephalitis B in our country,is an acute inflammatory disease of central nervous system caused by Japanese encephalitis virus(JEV),a mosquito-borne Flavivirus and a leading cause of epidemic encephalitis in Asia.As a neurotropic virus,JEV can effectively cross the blood-brain barrier(BBB)to cause acute viral encephalitis syndrome such as fever,coma,seizures,paralysis and even death in humans.While high morbidity and mortality caused by JEV infection,there are no specific and effective therapy available for controlling encephalitis.The neurotropism of JEV is the key event for pathogenesis.After infection of neurons,massive replication of JEV will directly induce neuronal necrosis and death.Meanwhile,immune activation in brain will be triggered by infection.Excessive immune activation and pathological damage of neurons are the key to the death and neurological sequelae.Since viruses are obligate intracellular parasites,it is no wonder that their replication depends heavily on host cell to obtain the energy and biological macromolecules.Therefore,viruses will inevitably evolve a plethora of strategies to reshape host metabolism in order to optimize its replication.Although anabolic phenotype conferred by virus infection like up-regulated of nutrients consumption,increased nucleotide and lipid biosynthesis is usually similar in coping with the high viral needs for proliferative infection,diverse viruses adopt distinct tactics to cope with different host cells and then lead to dissimilar metabolic landscape.Currently,whether or how JEV interact with host cell in metabolic facet during infection is still unknown.Hence,revealing the metabolic reprogramming of neuron cells caused by JEV infection and characterizing intracephalic metabolic landscape during JE are of great significance to clarify the pathogenic mechanism of JEV infection and provide novel target for clinical therapy.ObjectiveThis study takes JEV-infected neurons and JEV-challenged mice model as research objects to reveal the cellular metabolic reprogramming caused by JEV infection and characterize intracephalic metabolic profiles during JE.Our research aimed to clarify the interplay between virus and host cells in metabolic facet and unravel underlying mechanism associated with metabolism reprogramming.We hope that our research could help lay a theoretical foundation for the pathogenic mechanism of JEV infection,and provide a potential therapeutic target for the clinical treatment of JEV infection.Methods and ResultsMetabolomics and transcriptomics were integrated employed to characterize metabolic reprogramming in Neuro2a cell line infected with JEV and then,metabolomics was used to describe the metabolic profiles in brain during JE development in JEV-challenged mice model.Metabolic features both in JEV-infected neurons and brains with JE were systematically characterized and relevant mechanism associated with metabolic reshaping was identified.A preliminary intervention by targeting metabolism was carried out in JEV-challenged mice model in order to exploit promising antiviral strategies.1.JEV infection drastically rewires cellular metabolism and the underlying mechanism might be the activation of AMPK signaling pathway.The neurotropism of JEV is the key event for pathogenesis.In order to clarify the metabolic interplay between JEV and neuron cells,integrated metabolomics and transcriptomics analysis was employed in JEV-infected Neuro2a cell line.The results showed that JEV infection induced a drastically metabolic remodeling in Neuro2a cell line.Increase glycolysis and its branched pentose phosphate pathway(PPP)flux and impaired oxidative phosphorylation(OXPHOS)in glucose utilization were generated to facilitate the biosynthesis of precursors needed for viral replication.In addition,catabolic patterns of both amino acid metabolism and lipid metabolism were created within 24 hpi to cope with the high anabolic demands of virion production and optimal virus propagation.Lipid synthesis was subsequently promoted to avoid depletion.Then,metabolic-transcriptomic regulatory network was performed.It is to be noted that lipid metabolism was highly aggregated in the overall network analysis and indicates the key biological function of lipid metabolism in flaviviruses propagation.To unravel the relevant mechanism involved in host cellular metabolic reprogramming,western blot was performed and the phosphorylation level of AMPK was detected.The results showed that the phosphorylation level of AMPK was increased in the early stage of JEV infection and then decreased gradually.Transfection of different plasmids encoding virous viral proteins were further performed to clarify which viral protein was responsible for the activation of AMPK signaling pathway.The result suggested that several viral proteins,especially the non-structural protein NS3,were significantly involved in the activation of AMPK.The activation of AMPK signaling pathway usually exhibits catabolic features including the enhancement of glycolytic activity and the consumption of amino acids and fatty acids.These metabolic profiles were more favorable in maintaining the large amount of biomolecules and energy needed for JEV replication.2.JEV replication in neuron cells could be effectively inhibited by intervention of glucose metabolism.Based on the enhancement of glycolysis activity induced by JEV infection,the role of glucose metabolism in viral replication was systematically investigated.The results of glucose deprivation in neuro2a cell line and primary neurons infected with JEV suggested that the optimal replication of JEV was highly depended on glucose.Then,pharmacological inhibitions of both glycolysis pathway and its branched pentose phosphate pathway(PPP)were highlighted the importance of glycolysis and PPP for JEV replication in providing both ATP and nucleotides precursors.These results indicate that glucose metabolism,both glycolysis and PPP are indispensable for the optimal propagation of JEVAmino acid metabolism,is closely related with glucose metabolism.Thus,the role of glutamine(Gln),as a key intermediate for glucose,amino acid and nucleotide metabolism,in JEV infection was then investigated.The result of Gln deprivation in JEV-infected Neuro2a and primary neurons showed a significant enhancement of JEV infection under Gln-deprived condition.Meanwhile,exogenous addition Gln and 2-oxoglutarate(AKG),the intermediate connects Gln and tricarboxylic acid(TCA)cycle,exhibit inhibitory effect on JEV replication.siRNA was further employed to interfere with glutamate dehydrogenase 1(key enzyme catalyzes glutamate to AKG)and oxoglutarate dehydrogenase(key enzyme catalyzes the conversion of AKG to succiny-CoA in TCA cycle).The results showed that JEV replication was significantly enhanced while blocking the pathway of AKG generated from Gln and its utilization in TCA cycle.Thus,the axis of Gln-glutamate(Glu)-AKG played a corresponding role in inhibiting JEV replication.The distinct role of glucose and Gln in JEV propagation might associated with the activity of AMPK signaling pathway.3.Characterization of metabolic landscape in brain during JE development and a preliminary intervention targeting metabolismAlthough neuron cells are the most important target cell type for JEV infection,the replication of virus in neuron cells is usually an initiating factor for triggering inflammatory and JE occurrence.With the massive necrosis of neuron cells and the excessive activation of glia,brain tissue damage caused by severe inflammatory storm is the key event to death and neurological sequelae.Therefore,depicting the metabolic landscape of brain during the occurrence and development of JE and describing the complex metabolic changes caused by the interaction between virus and multiple cell types in the brain are important for revealing pathogenesis of infection,and providing potential therapeutic target.JEV-infected mice model was constructed and the brain tissues of mice at different stage of JE were employed for metabolomics analysis.The results suggested that the anabolism in the brain was enhanced,and the most of the metabolites,including amino acids and lipids,had increased in varying degrees.With the progression of JE,a large amount of metabolites in brain showed a decreasing trend compared with early stage.The possible reason might be that anabolism of brain was created both by viral replication and immune activation.With the progression,massive necrosis of neuron cells and over-activated immune lead to the decompensation of anabolism,and then,the decrease trend of most metabolites.Glutathione metabolism,which plays a key role in the maintenance of cellular redox homeostasis,was promoted in the early stage of JE to respond to extensive oxidative stress after JE occurrence,and then,with the consumption gradually reduced.Some metabolites such as N-Acetyl-aspartate and phosphocreatine,usually used as indicator for neural function and energy metabolism,were significantly decreased with the progression of JE and represent the severe damage in brain.Based on the significant antiviral performance of Gln at the cellular level,and its reduction level in brain metabolomics analysis,Gln was used as a preliminary intervention in JEV-infected mice model to determine its therapeutic effect.Through daily Gln intraperitoneal administration in mice infected with JEV from day 1 to day 5,the median survival time was prolonged in the high-dose challenged mice model and,the survival rate was improved in the low-dose challenged mice model.This preliminary intervention by targeting metabolism suggesting its feasibility as a therapeutical attempt.ConclusionIn summary,this study is the first to systematically describe the metabolic reprogramming of neuron cells after JEV infection and,depict metabolic landscape of brain in mice model infected with JEV.The underlying mechanism for this created metabolic reprogramming might be the activation of AMPK signaling pathway induced by JEV viral particles and related viral proteins.Through the deprivation of glucose and series pharmacological interventions of glycolysis and PPP in JEV-infected neuron cells,glucose metabolism,both glycolysis and PPP were demonstrated to be indispensable for the optimal propagation of JEV Gln-Glu-AKG axis exerts a significantly inhibitory effect on JEV replication and this inhibitory effect could be offset by blocking the pathway of AKG generated from Gln and its utilization in TCA cycle.Then,Gln was used as a preliminary intervention and exhibited beneficial effect in JEV-infected mice model.This work provides further insights into the pathogenic mechanisms of JEV infection in metabolic field and provides promising antiviral strategies targeting metabolism.