The Role Of Lipid Metabolism In Replication Of EV71

Background and ObjectsHand,foot and mouth disease(HFMD)is a common infectious disease that mainly infects the healthy of children under the age of five.Enterovirus 71(EV71)is one of the main pathogens that cause hand,foot and mouth disease.The hand,foot and mouth disease that EV71 causes can develop into severe neurological disease with complications such as myocarditis,pulmonary edema,aseptic meningitis,rapid development and high fatality rate.EV71 vaccine was launched in 2016.Although it has preventive effect on severe HFMD caused by EV71,the mechanism of severe HFMD caused by EV71 infection is still unclear.The study of EV71 infection can further explore the pathogenesis of HFMD caused by EV71,thereby helping to solve important and urgent clinical treatment problems.Studies have shown that EV71 infection can cause changes in intracellular substances and biochemical levels.The infection of most viruses can lead to changes in endoplasmic reticulum function and metabolic level,and the replication of viruses can be affected by intervening in these changes.This can provide an important basis for exploring the pathogenesis of HFMD caused by EV71 infection.However,these alterations have been rarely studied in EV71 infection.The endoplasmic reticulum is an important organelle in the cell.When the cell is stimulated by various external factors,the cellular homeostasis is broken and the endoplasmic reticulum produces stress.Endoplasmic reticulum stress is a cellular self-protection mechanism that triggers an unfolded protein response that helps clear cells of unfolded and misfolded proteins,thereby restoring ER homeostasis.It has been shown that infection of host cells with EV71 can induce endoplasmic reticulum stress.The endoplasmic reticulum is composed of a large number of membrane structures,and its structure and function are affected by the level of lipid metabolism in cells.Lipid metabolism plays an important role in the physiological process of normal cell growth,development and differentiation.Many studies have shown that some viruses can manipulate lipid metabolism in cells to promote their own replication after infecting cells.Poliovirus(PV)infection can cause changes in lipid metabolism in host cells.However,what role lipid metabolism and endoplasmic reticulum stress play in EV71 infection and how to play these roles deserve further exploration and research.In conclusion,exploring the interaction of EV71 with host cells may provide an important basis for the discovery of various drug targets for the treatment of viruses.Methods1.ER stress and EV71 replicationqPCR,Western blot and confocal microscopy were used to verify that EV71 virus with high multiplicity of infection(MOI=100)induces ER stress in human umbilical vein endothelial cells(HUVEC);After HUVECs were treated with endoplasmic reticulum stress inhibitor PKR IN and activator Tunicamycin,the replication level of EV71,the change of virus titer and the changes of lipid metabolism genes and proteins were observed.2.Lipid metabolism and EV71 replicationVerify that EV71 replication can cause changes in lipid metabolism.HUVECs were treated with lipid metabolism inhibitors TOFA,C75 or activator OA,and qPCR and Western blot techniques were used to study EV71 replication and changes in lipid metabolism genes and proteins.Confocal microscopy and electron microscopy was used to study lipid droplet changes.3.The role of lipid β-oxidation in the replication of EV71The β-oxidation inhibitor Etomoxir was used to further explore the effect of β-oxidation on EV71 replication,and then the siRNA of the β-oxidation rate-limiting enzyme CPT1 was designed to silence CPT1 and detect EV71 mRNA and protein level change.Cells were treated with the sugar metabolism inhibitor KAN0438757,and the cells were cultured in DMEM medium without sugar or sugar,and the changes of intracellular pyruvate were detected to observe the changes of EV71 replication level and titer,and to explore the effect of sugar metabolism and lipid metabolism on EV71 replication.Results1.EV71 at high multiplicity of infection(MOI=100)can induce ER stress in HUVEC cells,and inhibition(PKR IN)or activation(Tunicamycin)ER stress can inhibit EV71 replication,release and lipid metabolism.2.EV71 with high multiplicity of infection(MOI=100)can cause changes in lipid metabolism genes ACC,FASN,PLIN2 and PLIN2 proteins and lipid droplets in HUVEC cells;lipid metabolism inhibitors TOFA and C75 inhibit lipid metabolism.The number of polar and neutral lipid droplets was reduced,and EV71 replication was inhibited and released;lipid metabolism activator OA activated lipid metabolism,increased the number of polar and neutral lipid droplets,and promoted EV71 replication and release.3.The β-oxidation inhibitor Etomoxir inhibited the level of lipid metabolism,reduced polar and neutral lipid droplets,and inhibited the replication and release of EV71;after knockdown of CPT1 by siRNA,the expression of CPT1 mRNA and CPT1 protein decreased,and the level of EV71 replication decreased.After cells cultured in sugarfree DMEM,compared with the high-glucose group,the replication level of EV71 was reduced,and the virus release was reduced;the glucose metabolism inhibitor KAN043 8757 reduced the intracellular pyruvate content,inhibited glucose metabolism,and decreased lipid metabolism.The number of polar and neutral lipid droplets decreased,the replication level of EV71 decreased and the virus release decreased.TOFA and KAN043 8757 could all reduce the content of pyruvate in the pyruvate assay.ConclusionThe β-oxidation of lipids provides the main energy for EV71 virus replication.Endoplasmic reticulum stress is a concomitant change in cellular stress during EV71 virus replication,and has no restrictive effect on EV71 virus replication.