The Role And The Underlying Mechanisms Of IRG1 In Viral Replication

IRG1 is a metabolic enzyme located in mitochondria.It converts cis-aconitonic,an intermediate in tricarboxylic acid cycle,into itaconate.Although itaconate does not belong to the main line circulation of TCA,it affects the whole mitochondrial metabolic state and induces a series of physiological and biochemical changes.Itaconate has strong electrophilic properties due to its special chemical structure.It makes itaconate combine with cysteine,glutathione and other targets to form special chemical modification,then perform some regulatory activities,often resulting in the inactivation of functional elements.In recent years,IRG1-itaconate has been found to play important roles in inflammatory regulation,pathogen infection and metabolic diseases.The roles of itaconate in viral infection have rarely been reported.We speculated that the changes in metabolic state and gene expression profile caused by IRG1-itaconate will profoundly affect the relationship between host and virus.Therefore,we conducted the following study with the purpose of elucidation of the relationship between IRG1-itaconate and virus.Part ?.IRG1 promotes VSV replicationIn the previous transcriptome study,we found that IRG1 is the most induced metabolic gene upon viral infection.In the subsequent study,we found that IRG1 had regulatory effect on virus replication and carried out a mechanism study.Firstly,we verified whether IRG1 is a virus induced gene.We found that IRG1 was rapidly and strongly induced by various viral infections.Then we explored the function of IRG1 in viral infection by RNA interference technology and IRG1 knockout cells,and found that IRG1 could promote VSV replication through type I interferon independent manner.IRG1 deletion could significantly reduce VSV RNA in cells and titers in supernatants accompanied lower type I interferon expression.In VSV infection model,VSV RNA and titers in organs from IRG1 knockout mice were lower than those from wild-type mice,and cytokines in serum were also lower.Furthermore,interfering IRG1 in IRF3 knockout cells could still inhibit VSV replication.Part ?.IRG1 promotes VSV replication through the metabolite itaconateWe investigated whether itaconate was the cause of IRG1 promoting VSV replication.We used itaconate derivatives DMI and OI to study the function of itaconate.Firstly,we found that excessive DMI or OI treatment could eliminate the different VSV replication between the IRG1 interference group and the control group.And the same results were obtained in BMDMs and MEF cells with IRG1 deletion.Further studies demonstrated that the effects of DMI and OI on VSV replication were independent of type I interferon and downstream pathways.It was concluded from that the same promoting effect also existed in IRF3 or IFNAR1 deficient cells.In VSV infection model,pretreatment with DMI or OI could significantly increase VSV RNA in organs and type I interferon in serum.In addition,we also found that DMI and OI promoted endocytosis and intracellular replication during VSV infection.DMI and OI treatment promoted the entry of VSV for 1 hour,while IRG1 deletion decreased the entry.We transfected viral RNA into cells to bypass viral endocytosis,and DMI and OI still increased intracellular VSV RNA.In the same infection mode,viral RNA in IRG1-deficient cells were decreased.The data indicate that IRG1-itaconate axis acts on multiplate stages of VSV replication.Part ?.The mechanisms of IRG1-itaconate axis promoting VSV replicationWe studied the potential mechanisms of IRG1-itaconate axis promoting VSV replication.According to the previous reports,IRG1-itaconate axis functions by inhibiting SDH,promoting Nrf2-mediated gene transcription and regulating ROS production.We first explored and eliminated the above mechanisms.For SDH,we found that treatment with DMM,a classical inhibitor of SDH,had no significant effect on VSV replication in vitro and in vivo.Moreover,DMM did not affect the enhancing effect of DMI and OI on VSV replication as well.So we believe that they do not function through SDH.For Nrf2,interfering Nrf2 had no effect on the effects of DMI and OI promoting VSV replication.Therefore,we speculated that IRG1-itaconate did not play an effect through Nrf2.For ROS,our results demonstrated that DMI and OI treatment could significantly inhibit the production of ROS.Further study found that the effects of DMI and OI on VSV were not affected after ROS clearance in cells.So we believed that IRG1-itaconate did not play an effect through ROS.In conclusion,IRG1 did not promote VSV replication through the above three ways.In order to find out the potential mechanism of IRG1-itaconate axis promoting VSV replication,we conducted RNA-seq on IRG1 knockout and control cells.A large number of genes with reduced expression were enriched in oxidative phosphorylation related gene sets.The gene expressions were increased with treatment of DMI and OI.Meanwhile,ATP content in cells was also increased.ATP inhibitor treatment could partially reverse the effects of DMI and OI.Interference of related transcription factors had the same effect.It suggests that the increased energy metabolism might play an assistant role in the effect of IRG1-itaconate axis but not the key factor.On the other hand,we investigated whether lipid metabolism played a role in IRG1-itaconate axis promoting VSV replication.Firstly,inhibiting the transcription of lipid metabolism related genes could weaken the effects of DMI and OI.Inhibiting the synthesis of cholesterol almost blocked the effect of DMI and OI on VSV replication,so we focused on the pathway.Further research revealed that GGDP could rescue the effect of DMI and OI when the synthesis of cholesterol was blocked.It suggested that DMI and OI acted downstream of GGDP.GGDP could mediate prenylation and promote viral replication.Treatment with prenylation inhibitors blocked the effect of DMI and OI on VSV replication.This indicated that the effects of DMI and OI were largely dependent of prenylation.And we found that DMI and OI could increased the expression of genes coding prenylation related enzymes.Furthermore,itaconate facilitated the translocation of the classic target of prenylation,small GTPases,from cytoplasm to cell membrane.In conclusion,IRG1 induced by viral infection produced a mountain of itaconate to promoted VSV replication by up-regulating of oxidative phosphorylation related and prenylation related genes expression.This study provided a new understanding of the role and mechanism of IRG1-itaconate axis in viral infection.It will provide new targets and ideas for the treatment of clinically related diseases.