Effects And Mechanisms Of Lactate Dehydrogenase B And Autophagy Receptor NDP52 During Classical Swine Fever Virus Infection

Classical swine fever（CSF）is a severe infectious disease caused by classical swine fever virus（CSFV）,which causes high fever,hemorrhage,and organ necrosis in pig herds.At the same time,mild swine fever causes immune suppression,which poses a huge challenge for CSFV purification and prevention.In recent years,researches on the pathogenesis of CSFV have been continuously deepened,and studies have shown that CSFV infection inhibits the production of interferon and apoptosis in host cells to achieve continuous infection of philophils,and cause a large number of virus replication.However,the pathogenesis of CSF and the mechanism of its immune dysfunction are not fully clear.Therefore,studying the pathogenesis of CSF,especially the immune escape mechanism of CSFV has great significance for the prevention and control of CSF.Lactate dehydrogenase B（LDHB）is a key regulatory enzyme in the process of glycolysis metabolism,which affects the maturation of autophagosome to regulate autophagy.Autophagy is an internal balancing mechanism for maintaining homeostasis in eukaryotic cells.After receiving an autophagy induction signal,many components in the cytoplasm,including protein aggregates,damaged organelles,and foreign invading pathogens,are wrapped in vesicles and then closed into a closed spherical autophagosome Autophagy was initially considered to be non-selective,but recent studies have found that autophagy can also be selective.The most important feature of the selective autophagy pathway is the involvement of autophagy receptors that recognize and transport autophagic substrates,thereby regulating autophagy substrate degradation under very precise dynamic control.Previous research in our laboratory has showed that CSFV infection not only induce autophagy,but also use autophagy to promote the virus replication.Based on our previous finding that CSFV enhances autophagy for its replication in vitro,in the present study we perform two different studies related autophagy.On the one hand we studied the relationship between CSFV and LDHB,and revealed the effects and mechanisms of LDHB on CSFV replication through mitophagy;on the other hand,we studied the mechanism and function of the autophagy receptor NDP52 in CSFV infection,providing a new theory for the pathogenic and immune escape mechanisms of CSFV.LDHB inhibition activates NF-κB pathway,facilitating the progression of CSFV infection through mitophagy.Previous studies in our laboratory have shown that CSFV infection reshapes the glycolytic metabolism pathway processes and alters the production of metabolites.In order to verify the role of LDHB in the regulation of glycolysis by CSFV,the changes of lactic acid and pyruvate in the serum of Tibetan miniature pigs or PK-15 cell supernatant were detected after CSFV infection.We found that CSFV infection increased the lactic acid content and decreased pyruvate content.However,LDHB inhibition changed the tendency of lactic acid increase and pyruvate decrease in PK-15 cells by infected with CSFV,indicating that LDHB plays an important role in the regulation of CSF on lactic acid and pyruvate.The CSFV non-structural protein NS3 is essential in viral replication.In order to study the interaction between LDHB and CSFV,we used yeast two-hybrid system,GST-Pull down co-immunoprecipitation and laser confocal microscopy to verify the CSFV replication protein NS3 interacts with LDHB protein.Subsequently,the expression of LDHB in liver,kidney,spleen,and tonsil of CSFV-infected pigs was detected by q RT-PCR and immunohistochemistry.It was found that CSFV infection can significantly reduce LDHB expression in above organs.Moreover,the expression of LDHB was also inhibited in CSFV infection PK-15 and 3D4/2 cells.Recent studies have shown that tumor cell mitochondrial metabolism is closely related to glycolysis metabolism,and LDHB is located on the outer mitochondrial membrane.In order to study the relationship between LDHB and CSFV replication,we used LDHB-specific interfering RNA to inhibit the LDHB gene expression in PK-15 and 3D4/2cells.At the same time,we used transmission electron microscopy to observe the mitochondrial morphology;used western blot to detect the expression of internal and external model proteins of mitochondrial autophagy and the ubiquitination of MFN2protein;used laser confocal microscopy to test the co-localization of Mito-Tracker,GFP-LC3 and CD63 and to detect the fluorescence change with Mito-GFP-RFP dual fluorescent plasmid.The results showed that a large number of mitochondria wrapped in membranous structures appeared in the cells after the inhibition of LDHB,and the length of the mitochondria became shorter;western blot results showed that the expression of molecules such as TOM20 and VDAC was reduced,and the ubiquitination of MFN2protein increases;Laser confocal microscopy results found that the intracellular green fluorescence decreased and the co-localization of Mito-Tracker,GFP-LC3 and CD63 was increased after the inhibition of LDHB.The above results indicated that LDHB inhibition promotes completely cell mitophagy.To investigate the effect of mitophagy induced by LDHB inhibition on innate immunity,we first examine the effect of LDHB on the NF-k B signaling pathway and cytokines release.Using double luciferase experiments,it was found that LDHB inhibition activates the NF-κB signaling pathway.Western blot results found that inhibition of LDHB promotes the expression of NF-κB signaling pathway related proteins P65,IκB-A,p-IκB-A,IκB-Ras-2,but CSFV infection reduces the expression of NF-κB signaling pathway-related proteins that induced by LDHB inhibition,indicating that CSFV infection attenuates NF-κB signaling pathway.Moreover,LDHB inhibition obviously enhances the transcription and release levels of IFNα,IFNβand TNF cytokines,but CSFV infection inhibits the release of the above cytokines to promote the virus replication.To find out whether LDHB acts on the activation of NF-κB signaling pathway through mitophagy,we selected mitophagy interference plasmid sh Parkin and mitophagy activator CCCP to treat PK-15 and 3D4/2cells,and then transfected with LDHB-specific interference RNA,it was found that inhibition LDHB activating NF-κB signaling pathway through mitophagy.Further research found that CCCP-induced mitophagy inhibits the production of cytokines,and sh Parkin interference plasmids to promote the production of cytokines.To investigate whether LDHB affects CSFV replication through mitophagy or NF-κB signaling pathway,we used specific interfering RNA to target knockdown of LDHB protein expression,or transiently transfect Flag-LDHB plasmid into PK-15 and 3D4/2 cells to overexpress LDHB protein,Then,PK-15 and 3D4/2 cells were infected with CSFV with MOI=1,and Western blot was used to detect LDHB interference and overexpression effects and the expression of CSFV N^pro protein.It was found that inhibiting LDHB can promote the expression of CSFV N^pro protein,while overexpress LDHB has the contrary effect,it shows that inhibiting LDHB has the effect of promoting CSFV replication,while overexpressing LDHB reduces CSFV replication.At the same time,we detected the CSFV m RNA copy number and virus titer in the cell supernatant 24 hours after CSFV infection.The results showed that inhibition of LDHB up-regulated the CSFV m RNA copy number and virus titer in the cell supernatant,while overexpression of Flag-LDHB inhibited CSFV replication.The above results indicate that LDHB has a negative regulatory effect on CSFV replication.To further study the mechanism by which LDHB inhibits CSFV replication,PK-15 and 3D4/2 cells were treated with mitophagy activator CCCP,mitophagy inhibitory interference plasmid sh Parkin and NF-k B signaling pathway inhibitor BAY,and then transfected with LDHB specific Transfected with sexual interfering RNA,and infected cells with CSFV with MOI=1.after 24 h transfection,using Western blot to detect the expression of LDHB and N^pro protein or q RT-PCR to detect the relative expression of CSFV NS5B m RNA,it was found that CCCP can cooperate LDHB interfering RNA promotes the expression of CSFV N^pro protein and the relative expression of NS5B m RNA,but sh Parkin antagonizes the expression of CSFV N^pro by LDHB interfering RNA.The effect of BAY on the expression of CSFV N^pro protein and the relative expression of NS5B m RNA is not obvious,indicating the inhibition of LDHB mainly acts on the replication of CSFV through mitophagy.Studies have shown that CSFV infection can inhibit apoptosis and promote self-replication.To detect the effects of LDHB on apoptosis,we examined the apoptosis of PK-15 and 3D4/2 cells after LDHB inhibition.Western blot results showed that the expression of apoptosis-related molecules such as PARP,caspase-3,and caspase-9 was decreased.Flow cytometry results showed that inhibition of LDHB reduced the proportion of double positive apoptotic cells,and overexpression of LDHB increased the proportion of double positive apoptotic cells,suggesting LDHB may promote CSFV replication through apoptosis.The role and mechanism of NDP52 in CSFV infections.NDP52 is an autophagy receptor and plays important role in the infection of pathogenic microorganisms.Our previous studies found that CSFV infection induced mitophagy and use autophagosome as a place for self-replication,but the mechanism by which CSFV enters autophagosome is unclear.In order to study the interaction between the autophagy receptor NDP52 and CSFV,the expression and protein modification levels of NDP52 after CSFV infection in PK-15cells were tested.It was found that CSFV infection inhibited the expression,transcription,ubiquitination and SUMO2-4 modification of NDP52 protein in PK-15 cells.Since the autophagy receptor protein ubiquitination modification is closely related to the PINK1-Parkin pathway,in order to verify whether the CSFV modification of NDP52protein is regulated by the PINK1-Parkin pathway,the effect of CSFV infection on the parkin protein was first tested,and it was found that CSFV infection can promote parkin protein expression and ubiquitination.Further transfection of sh Parkin interfering plasmid into PK-15 cells and infection with CSFV revealed that inhibition of the PINK1-Parkin pathway up-regulates the expression of modified NDP52 protein,indicating that CSFV regulates the protein modification of NDP52 through the PINK1-Parkin pathway.In order to study the effect of NDP52 on CSFV replication,specific interfering RNA was used to suppress the expression of NDP52.It was found that the virus titer and virus copy number of CSFV in PK-15 cells were reduced,and the expression of viral protein N^pro was reduced,indicating that NDP52 promotes CSFV replication.Autophagy receptors mediate the occurrence of selective autophagy.In order to study the effect of NDP52 on autophagy,we used confocal laser to observe the change of fluorescence intensity in the GFP-LC3 and GFP-RFP-LC3 autophagy dual-fluorescence reports and western blot to detect the autophagy marker molecules expression in the PK-15cells with NDP52 inhibition.The results showed that the fluorescence intensity of GFP-LC3 decreased;the proportion of GFP fluorescence in GFP-RFP-LC3 autophagy double fluorescence increased;the expression of autophagy proteins such as LC3 and Becn I decreased,while the expression of TOM20 and VDAC-1 increased.The above results indicate that NDP52 inhibition decreases the occurrence of autophagy.CD63 is a lysosomal marker molecule.Studies have shown that CD63 interacted with the CSFV E2 protein.After inhibiting NDP52,the expression of CD63 protein and the binding to E2 protein was decreased in the cytoplasm.At the same time,the binding of parkin and E2 protein also decreased.It suggested that inhibition of NDP52 can inhibit the entry of CSFV into autophagosome by reducing the binding of CD63 and E2,thereby inhibiting CSFV replication.Further research found that NDP52 inhibition activates NF-κB signaling pathway and promotes cytokine transcription.In summary,we found that LDHB inhibition activates NF-κB signaling and promotes CSFV replication through mitophagy.Inhibiting NDP52 affects the binding of CSFV E2protein to CD63 and parkin molecules and reduces CSFV replication levels.