Emerging Roles Of Free Fatty Acids Metabolism And Lipid Droplets On Classical Swine Fever Virus Infection

Classical swine fever(CSF)is a serious porcine disease driven by the CSF virus(CSFV)has brought substantial economic losses to the world pig industry.Currently,treatment options for CSF are still limited;instead,prevention with vaccines against CSFV is usually used.However,under immune selection pressure,CSFV has evolved and developed mechanisms that escape the host immune response,resulting in an outbreak of CSF or establishing persistent infection in an immune flock.Although many studies have investigated the interaction mechanism between CSFV and the host,the pathogenesis and immune escape mechanism of CSFV still remain unclear.It is still necessary to explore the pathogenic mechanisms of CSFV in order to develop specific drugs and vaccines for effective CSF prevention,control,and eradication.Lipids are the important component of cells and internal organelle membranes,which not only regulate various cellular life processes,but also play an important role in virus infection.As an intracellular parasitic microorganism,viruses need lipid biogenesis participation in various steps of infection,and also alter lipid metabolism and provide favorable conditions for their replication.In recent years,the changes of lipid composition,lipid distribution and lipid content in cell membranes and cells,and abnormal lipid metabolism have also attracted more and more attention and research as potential pathogenesis of various viral infectious diseases.Our previous work has confirmed that CSFV rebuild cellular metabolic programs in vitro and in viro,thus aiding viral replication.However,the mechanisms of lipid metabolism in the pathogenesis of CSFV infection and host antiviral immunity remain unclear.In view of these,the current study aims to reveal molecular mechanisms of pathogenicity and immune regulation of lipid metabolism in CSFV infection for the first time.At first,we performed a serum lipidomics analysis of piglets infected with CSFV.And based on this,we selected the appropriate cells as the model of CSFV infection in vitro,and systematically analyzed the mechanism of free fatty acid,fatty acid β-oxidation,lipid droplet and lipophagy in CSFV infection from different levels and perspectives,so as to reveal the pathogenesis of CSFV from a new perspective,and provide the necessary scientific theoretical basis for the prevention,control,and eradication of CSF.Serum lipidomics analysis of CSFV infection in piglets and emerging role of free fatty acids on virus infection.In the present study,for the fist time,we performed serum lipidomics analysis of piglets infected with CSFV based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS),and identified 167 differentially expressed lipid metabolites.Interestingly,FFAs accumulated significantly in these metabolites,accompanied by an increase in sphingolipids and a decrease in glycerolipids and glycerophospholipids,suggesting that CSFV infection markedly changed the serum lipid metabolism of piglets.FFAs are the principal constituents of many complex lipids and are essential substrates for energy metabolism.Based on this,we focused on whether FFAs play a prominent role in CSFV infection.We found that CSFV infection induced FFAs accumulation in vivo and in vitro,which is due to increased fatty acid biosynthesis.Meanwhile,we discovered that alteration of cellular FFAs accumulation by a mixture of FFAs or inhibitors of fatty acid biosynthesis affects progeny virus production in vitro.Furthermore,in the absence of glucose or glutamine,CSFV still has replication capacity,which is significantly reduced with the addition of fatty acid beta oxidation inhibitors,suggesting that the process of FFAs enter the mitochondria for beta oxidation to produce ATP is necessary for virus replication.Finally,we demonstrated CSFV induced FFAs accumulation results in impaired type I IFN signaling-mediated antiviral responses by down-regulating RIG-I-like receptors(RLRs)signaling molecules,which may represent a mechanism of CSFV replication.Taken together,these findings provide the first data on lipid metabolites during CSFV infection and reveal a new view that CSFV infection requires FFAs to enhance viral replication.CSFV infection activated AMPK mediated autophagy regulates fatty acid βoxidation.FAO is an important pathway of intracellular FFAs decomposition for energy,and is closely related to virus infection.In order to reveal the regulation and mechanism of FAO in CSFV infection,PK-15 and 3D4/2 cells were infected by CSFV virulent "Shimen" strain.The FAO rate and the expression of FAO key proteins of CSFV infected cells were detected.The results showed that CSFV infection inhibited FAO in the early stage and promoted FAO in the later stage.To analyze the mechanism of CSFV infection regulating FAO,we detected the AMPK activity of CSFV infected target cells 48 hours later,and used the activator AICAR or inhibitor dorsomorphin to regulate the AMPK activity of cells,and analyzed the effect of AMPK activity on the FAO of CSFV infected cells.Our results showed that CSFV infection induced AMPK activation and regulated the host cell FAO.AMPK is not only a cell energy receptor,but also an important factor to induce autophagy.To further study the mechanism of CSFV infection induced AMPK activation regulates autophagy,cells were treated with AMPK regulator,and the expression level of autophagy marker molecules p62,LC3,BECN1 and ATG5 were detected by Western blot.The results suggested that CSFV-activated AMPK induces autophagy.In order to further study the role of autophagy in the regulation of FAO in CSFV infected cells,we used activator rapamycin and inhibitor 3-MA to regulate autophagy activity,which proved that CSFV infection induced autophagy regulates FAO.Moreover,we used the active regulator of autophagy and AMPK to treat cells.The results showed that CSFV-activated AMPK induces autophagy to regulates FAO.In addition,we used etomoxir and ciprofibrate to control the rate of cell FAO,which proves that CSFV replication depends on FAO.More importantly,we also found that the promotion of FAO slowed down the inflammatory response of CSFV infected cells,while the inhibition of FAO was the opposite.Taken together,all these results showed that CSFV infection induced AMPK activation mediated autophagy regulation of FAO can slow down the inflammatory response of host cells,which may be an important mechanism for CSFV to achieve persistent infection and immune escape.CSFV induces lipid droplets accumulation and lipophagy for optimal replication.Lipid droplets are multifunctional organelles that store neutral lipids in eukaryotic cells and play an important role in maintaining homeostasis of lipid metabolism.Previous studies have shown that lipid droplets were not only the platform of virus proliferation,but also the specific inducement of lipophagy provided favorable conditions for virus replication.In order to study the mechanism of lipid droplets and lipophagy in CSFV infection,HSC cells was selected as the model of CSFV infection in vitro for the first time.The growth characteristics of CSFV in HSC cells were analyzed by indirect immunofluorescence technique.The results showed that CSFV can infect HSC cells and complete proliferation and replication in cells.By detecting the transcription level of fatty acid metabolism related genes,intracellular FFAs and FAO rate in CSFV infected HSC cells at different time points,We found that CSFV infection resulted in FFAs accumulation and FAO abnormality in HSC cells.Then,we detected the number of lipid droplets,TG and CE content,the expression level of lipid droplet structural protein in CSFV infected HSC cells at different time points.The results showed that CSFV infection resulted in the accumulation of lipid droplets,while uv-csfv,which lost the ability of virus replication,had no significant effect on lipid droplets.Subsequently,we used triacsin C and oleic acid to regulate the lipid droplets content of HSC cells,and found that CSFV replication depended on lipid droplets.The co localization of lipid droplets and CSFV structural protein E2 in CSFV infected cells was further detected by laser confocal technique.The results showed that CSFV infection not only enhanced the fluorescence signal of lipid droplets,but also caused the co localization of lipid droplets and CSFV-E2 fluorescence signal,suggesting that lipid droplets can provide a platform for CSFV particle assembly.The decrease of the number of lipid droplets and the increase of FFAs in the later stage of CSFV infection suggest that the degradation of lipid droplets is induced by CSFV infection.Autophagy is one of the most important ways to degrade lipid droplets.To analyze the role of autophagy in the degradation of lipid droplets in CSFV infected cells,the expression of autophagy marker protein in CSFV infected cells were detected by western blot.Further,we also used rapamycin and 3-m A to regulate the autophagy activity of HSC cells,which proved that CSFV infection induced autophagy to degrade lipid droplets.The process of autophagy degrade lipid droplets is also called "lipophagy".In order to confirm that CSFV infection can induce lipophagocytosis in HSC cells,we used transmission electron microscopy to analyze the lipid droplets and autophagy like vesicles in CSFV infected cells,and found that lipophage-like structures with autophagosome-like vesicles contacting or encapsulating lipid droplets appeared in CSFV-infected HSC cells,which proved morphologically that CSFV infection induced lipophagy.In addition,the co location of lipid droplets and LC3 in CSFV-infected cells for 48 hours was analyzed by laser confocal technique,which further proved that CSFV infection induced lipophagy.Moreover,we use agonist Rapamycin and inhibitor 3-MA to regulate cell autophagy activity,and proved that CSFV infection induced lipophagy to promote the synthesis and oxidation of fatty acids.In summary,this study for the first time confirmed that CSFV infection induces lipid droplet accumulation and uses lipid droplets as a replication platform,and regulates lipid metabolism by inducing lipophagy to degrade lipid droplets,which may be an important mechanism for CSFV to achieve sustained infection.