Role Of Mitophagy And Autophagy On Classical Swine Fever Virus Infection And Metabolomics Of Classical Swine Fever Virus

Classical swine fever(CSF) is an acute, febrile, highly contagious disease caused by classical swine fever virus(CSFV), and is an OIE(World Organisation for Animal Health)-listed disease. CSF caused by high virulence CSFV infection is characterized by haemorrhagic syndrome and immunosuppression, while severe immunosuppression with no obvious appearance caused by strains of low to moderate virulence takes more difficulty on control and clearance of CSF. Monocytes and vascular endothelial cells are main targets for CSFV-infection in vivo, but it is curiously enough that immunosuppression is mainly caused by depletion of leukocyte populations including B and T cells. Previous report has showed that CSFV inhibits apoptosis and interferon synthesis during the establishment of long-term infection and replication in target host cells. Although many studies are doing the best to explore the competitive realtion between CSFV and host cells, the pathogenesis and immune dysfunction during CSF is still unclear.Therefore, it is essential to deeply clarify the pathogenesis and immune evasion mechanism of CSF for effectively controlling infection.As an evolutionarily conserved defence mechanism, autophagy plays an important role on virus infection. Recent study shows that lots of virus indudes mitophagy to clear up damaged mitochondria in host cells. In addition, autophagy is closely related to cell death,and cellular metabolism. Based on our previous finding that CSFV enhances autophagy for its replication in vitro, in the present study we perform three different studies related autophagy. Firstly, the mechanism of inhibition of apoptosis by CSFV through activation of mitochondrial fission and mitophagy was studied. Secondly, the relation of CSFV-infection and mitophagy was studied to explore the effect of mitophagy on CSFVreplication and apoptosis. Secondly, the relation of autophagy and apoptosis in the spleen of pigs infected by CSFV was analysed to explore the role of autophagy on splenic cell death. Thirdly, metabolomics approach was ultilzed for dynamicly and multiplely analysing effects on CSFV infection to understand the role of glucose, nucleotide,aminoacid and fatty acid metabolism on CSFV replication and immune evasion.CSFV induced mitophagy to inhibit apoptosis of infected cells. Many viral proteins can target to mitochondria through their mitochondrial localization sequences during virus infection. Damaged mitochondria caused by virus infection can be cleared up through mitophagy. To explore the mechanism of CSFV-induced mitophagy and the role of mitophagy on CSFV infection, PK-15 and 3D4/2 cells were both used and infected by CSFV. PK-15 cells are easily infected by CSFV, and 3D4/2 cells are alveolar macrophages which are representative of innate immune cells. We firstly tested changes of mitochondria mass in PK-15 and 3D4/2 cells at different hours post CSFV infection. The Weston blot results showed that mitochondrial mass obviously derceased after 36 h post infection. To prove that degradation of mitochondrial mass were related to mitophagy, we treated cells with 3-methlyadenine(3-MA) inhibiting the phagophore formation and Bafilomycin A1(Baf A1) inhibiting the activity of vacuolar-type H+-ATPase before and during CSFV infection. Both autophagy inhibitors can invert the decline of mitochondrial mass induced by CSFV infection. To confirm whether CSFV-induced mitophay, we examined the formation of mitophasomes in CSFV-infected PK-15 and 3D4/2 cells using transmission electron microscopy(TEM). The results showed that mitochondria were trapped by double membrane vesicles in CSFV-infected cells.To explore the mechanism of mitophagy induced by CSFV infection, purified mitochondrial of CSFV-infected cells were firstly prepared and then analysed by Weston blot. It showed that increased translocation of Pink1 and Parkin on purified mitochondria was detected. To assess the effect of Parkin translocation to mitochondria, ubiquitination of MFN2 was analyzed by Westonblot. The results showed that CSFV upregulated ubiquitination of MFN2 both in PK-15 and 3D4/2 cells. And the decline of MFN2 was associatd with its ubiquitination. To confirm mitochondria engulfed by atophagicphagophore was marked by Parkin, PK-15 and 3D4/2 cells transfected with GFP-LC3 plasmid were infected by CSFV and analyzed by confocal immunofluorescence assay. The data showed the mitohagosome conjunct with Parkin was trapping by EGFP-LC3 puncta.To confirm mitochondria in PK-15 and 3D4/2 cells infected by CSFV was finally delivered to lysosome, we utilizing a tandem-tagged m RFP-GFP plasmid encoding a mitochondrial targeting signal sequence, which was based on the differential stabilities of RFP and GFP in lysosomes to display mitophagy. It showed that cells infected by CSFV displaying more RFP fluorescence, which indicated the degration of mitochondria by lysosomes. To confirm the fusion of mitophagosome with lysosomes, merging of GFP-LC3, mitochondria and lysosomes in PK-15 and 3D4/2 cells was analysed by confocal microscopy. The images showed that mitochondria wrapped by LC3 puncta were connected with lysosomes in cells infected by CSFV. It proved the formation of mitophagolysome in CSFV-infected cells.Based on CSFV-induced mitophagy in the present study, we further observed the changes of mitochondria in CSFV-infected cells using confocal immunofluorescence assay.The results showed that CSFV caused fragmented mitochondria and increased level of mitochondrial translocation of Drp1. To check the functional effect of mitochondrial fission and mitophagy on the course of CSFV infection, we determined CSFV Npro expression, RNA replication and virus titers in the cells silenced endogenous Drp1 or Parkin by sh RNA knockdown experiments. The results explored that Drp1 or Parkin silencing decreased viral replication. To further check that inhibition of CSFV replication in the Drp1- or Parkin-silenced cells is a consequence of apoptosis. Cleaved-caspase-3 and cleaved-PARP was analyzed by Weston blot, and apoptosis of cells were funther confirmed by flow cytometry assay. The results showed that CSFV enhanced apoptosis of cells depleted of mitochondrial fission or mitophagy.CSFV caused atophagic death of partial T-area cells in the spleen. Spleen, where large numbers of CSFV particles reside, contains various kinds of macrophage and lymphocyte populations, so it is a very suitable organ for analyzing the relation of CSFV infection, autophagy and apoptosis in vivo. To detect distribution of CSFV antigen, theparaffin sections of spleen were stained with CSFV E2 antibody. The results showed that most of cells in the spleen of piglets were infected by CSFV. To clarify the changes of autophagy in the spleen of pigs infected by CSFV, we firstly evaluated the expression of LC3II/LC3 I and SQSTM1. Contrary to the increased levels of LC3II/LC3 I, SQSTM1 showed a decreased feature. It showed CSFV enhanced autophagy in the spleen. We further assessed the changes of autophagy related proteins including ATG5 and BECN1 by Weston blot. The results showed that CSFV raised expression of ATG5 and BECN1 in splenic cells. To examine the spatial distribution of autophagic cells in the spleen of pigs infected by CSFV, the paraffin sections were stained by LC3 II specific antibody. The data showed that CSFV caused LC3II-positive cells mainly distributing around splenic corpuscles.To confirm the involvement of apoptosis in the spleen of pigs infected by CSFV,further research was carried out. Firstly, the apoptotic frequency of dispersed splenic cells was analysed by flow cytometry. In order to confirm apoptotic signals were increased in the spleen of CSFV-infected piglets, hallmark proteins of apoptosis were analyzed by immunoblotting. Our results showed that levels of cleaved caspases-8, caspases-9,caspases-3 and PARP were enhanced in CSFV-infected spleen cells. To further observe the location of apoptotic cells, we stained apoptic cells using TUNEL method. Interestingly,most positive cells were scattered around splenic corpuscles in CSFV infected groups,which refered the relation of autophagy and apoptosis in the spleen of pigs infected by CSFV.To clarify whether autophagy were associated with apoptosis in the spleen of piglets inoculated with CSFV, frozen sections were double stained with LC3II-antibody and TUNEL. Quantitative analysis of confocal immunofluorescence image revealed that partial LC3II-positive cells(~40%) were stained with TUNEL in CSFV-infected groups. We further examined whether autophagy and apoptosis were directly caused by CSFV infection in the spleen. By staining CSFV E2 protein, confocal immunofluorescence of LC3 II and TUNEL merged with CSFV antigen were inspected. Convincingly, most apoptotic cells were not infected by CSFV in vivo. However, it was worth noting that asmall proportion of autophagic cells were not infected by CSFV and not all CSFV-infected cells were undergoing autophagy.Metabolomics of CSFV infection. Virus can disturb the changes of metabolisms in host cells resulting diseases. The relation of CSFV infection and changes of metabolisms has not been explored. In the present study, PK-15 and 3D4/2 cells were infected with CSFV and annalysed by metabolomics. In glucose metabolism, the results showed that CSFV caused decline of glucose, fructose-6-phosphate and glyceraldehyde-3phosphate during glycolysis in PK-15 cells, and decline of glyceraldehyde-3phosphate,phosphoenolpyruvate and pyruvate were detected in CSFV-infected 3D4/2 cells. This showed that CSFV increased the utilization of metabolisms in glycolysis, which pointed us that CSFV enhanced glycolysis in host cells. During tricarboxylic acid cycle(TCA), CSFV increased citric acid, isocitrate, 2-Ketoglutaric acid in PK-15 cells, but decreased them in3D4/2 cells. It showed that CSFV upregulated TCA to offer enough energy for viral replication, whereas CSFV inhibit TCA to limit the energy used for immune activity in3D4/2 cells.In nucleotide metabolism, we found that decrease level of glucose and ribulose-5-phosphate were dected during pentose phosphate pathway in PK-15 cells infected by CSFV. During conversion of pentose glucuronic acid, glucuronic acid, xylitol,D-xylulose and arabitol were decreased by CSFV infection in PK-15 cells. These data displayed that CSFV may upregulate pentose phosphate pathway and conversion of pentose glucuronic acid to produce more ribose for viral RNA replication. Moreover, low level of hypoxanthosine, inosincacid and guanine in PK-15 cells further proved acitivy of RNA synthesis was enhanced by CSFV. Meanwhile, decline of uridine 5- monophosphate in 3D4/2 cells hinted us that RNA synthesis were upregulated by CSFV infection.In aminoacid metabolism, CSFV inhibited alanine, aspartate and glutamate metabolism, alanine and aspartate metabolism, glycine, serine and threonine metabolism,valine, leucine and isoleucine degradation in PK-15 cells, whereas alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine degradation, beta-alanine metabolism and taurine and hypotaurine metabolism were activated by CSFV infection in 3D4/2 cells.The data proved that CSFV may enhance the production of proteins in PK-15 cells, but exerted a dcreased function in 3D4/2 cells.In fatty acid metabolism, our data explored that palmitelaidic acid and palmitoleic acid dcreased in PK-15 cells infected by CSFV, but palmitoleic acid in 3D4/2 cells was increased by CSFV infection. It proved that CSFV regulated fatty acid metabolism according to different cell types.