The Mechanism Of HDAC8 Autophagic Degradation Mediated By FMDV VP3 Protein During Virus Infection

Foot and mouth disease is an acute,highly contagious disease caused by foot and mouth disease virus.Every year,foot-and-mouth disease causes huge economic and social impact to many countries in the world.Deeply study the epidemic characteristics of the pathogen and the molecular mechanism of the interaction between the virus and the host that will provide a theoretical basis for the prevention and control of foot-and-mouth disease and vaccine research and development.In this study,we found that foot-and-mouth disease virus infection can induce the degradation of HDAC8 in host cells.Further studies showed that the structural protein VP3 of FMDV combined and promoted HDAC8 autophagy degradation.This study explored the new mechanism of FMDV VP3 protein to antagonize the innate immune response.The results are as follows.（1）HDAC8 inhibits the replication of FMDV.In order to investigate whether histone deacetylase regulates the replication of FMDV,the general inhibitor of histone deacetylase,class I histone deacetylase inhibitor and histone deacetylase 8 specific inhibitor were used.The results show that:In different cell lines,compared with the control group,the replication of FMDV increased in different degrees after treatment with the inhibitor,and the effect of HDAC8 specific inhibitor was the most obvious.To further explore the effect of HDAC8 on the replication of FMDV,HDAC8 knockout cell lines were constructed in BHK-21 and PK-15 cells using CRISPR/cas9 technology.From the three dimensions of protein,transcription and virus titer,HDAC8 knockout significantly promoted the replication of FMDV in different cell lines.（2）FMDV infection significant reduces the the expression of HDAC8 protein but not its transcription.In order to explore the effect of FMDV infection on the expression of HDAC8 protein or the transcription level,cells were inoculated into a 6-well plate with the same number of cells and infected with 0.1 MOI FMDV respectively,and the samples were collected at 0 h,1 h,3 h,5 h,7 h and 9 h.The results showed that the expression of HDAC8 protein was significantly decreased with FMDV infection in a time-dependent manner,but the transcription level of HDAC8 did not significantly change.In order to further explore the effect of different MOI FMDV on the HDAC8protein level,cells were infected with 0,0.01 MOI,0.05 MOI,0.10 MOI,0.25 MOI FMDV and collect samples after 8 hours of virus infection.The results showed that the expression level of HDAC8 protein significantly decreased with the increase of the dose of FMDV,which was dose dependent,but had no significant effect on the transcription level of HDAC8.（3）HDAC8 promotes the activation of innate immune response.In order to investigate whether HDAC8 is involved in the regulation of innate immune signaling pathway,PK-15-KOHDAC8 knockout cell lines and control cell lines were used to collect protein samples or RNA samples after infected with 0.1 MOI FMDV for 8 hours.The results showed that the phosphorylation of TBK1 and IRF3 decreased significantly after HDAC8protein was knocked out.In addition,overexpression of HDAC8 significantly potentiated the phosphorylation of TBK1 and IRF3 during FMDV infection and then weakened the viral replication.Real-time PCR was explored that the expression level ofβ-IFN,ISG54,CCL5,OAS,and TNF-αdecreased in HDAC8 knockout cells and significantly increased in HDAC8overexpression cells.Therefore,HDAC8 can inhibit the replication of FMDV by regulating the innate immune response and indicate that HDAC8 was involved in the innate immune signal pathway.（4）VP3 interacts and promotes HDAC8 degradation through AKT-m TOR-ATG5 pathwayFMDV contains 4 structural proteins and 8 non-structural proteins.In order to screen which protein responsible for degrading HDAC8,different FMDV gene plasmids were transfected into PK-15 cells and the protein samples were collected after 24 h with FMDV infection.The results showed that compared with control,transfection of VP3 and 3C^proplasmids showed a decreased expression of HDAC8.3C^prois a known protease to universally clease host proteins to facilitate FMDV replication,so we focused on VP3 protein for further study.In order to further explore whether the VP3 protein of different FMDV stains can degrade HDAC8,the VP3 protein of O,A and AsianⅠFMDV was gradient transfected or co-transfected with HA-HDAC8 plasmid into PK-15 cells.The results showed that VP3-induced HDAC8 degradation is conserved through all three FMDV serotypes.In order to further explore whether there is protein interaction and subcellular co-location between VP3 and HDAC8,the FLAG-VP3 plasmid,HA-HDAC8 plasmid and control plasmid were transfected into 293T cells respectively.The results showed that VP3 and HDAC8 had protein interaction and subcellular co-localization.For further exploration the key regions of VP3 involving in interaction or degradation of HDAC8,Co-transfer VP3 truncated plasmid with HA-HDAC8 into PK-15 cells or 293T cells.The results showed that the dagradation of HDAC8 was recovered when the 315-465 or 466-660 amino acids region of VP3 was deleted and only the 315-465 amino acids region of VP3 showed the interaction.To verify which degradation pathway is involved during FMDV infection,different inhibitors of degradation pathways were added to the culturing cells co-transfected with VP3 and HDAC8.The results showed that the addition of autophagy degradation pathway inhibitor 3-MA and CQ showed significant recovery of HDAC8 expression.In order to further explore whether VP3 protein can induce autophagy,the control plasmid and VP3 plasmid were transfected into PK-15 cells.The results showed that the expression of VP3 increased the protein level of LC3B-Ⅱ,the accumulation of fluorescent puncta of LC3B and more vesicles with electron microscopy.LC3B and P62 are two marker proteins of autophagy.In this study,we found that VP3 protein co-localization and interacts with LC3 and P62.In order to further explore the molecular mechanism about VP3promotes HDAC8 autophagic degradation,the control plasmid and VP3 plasmid were transfected into PK-15 cells.The results showed that compared with control,transfection of VP3 in PK-15cells inhibited the phosphorylation of AKT and m TOR,thus leading VP3 induced autophagy through AKT-m TOR signaling pathway.SC79,a specific AKT activator,was used to activate the AKT in the cytoplasm,inhibit AKT membrane translocation.When treat with SC79 the expression of HDAC8 was recovered in a dose-dependent manner and the phosphorylation of AKT and m TOR recovered to inhibit autophagy induced by VP3.ATG5 is a key node molecule in autophagy process.We found that through transfecting VP3 plasmid into ATG5 knockout cell line,VP3 could not induce autophagy and degrade the expression of HDAC8.In summary,we revealed for the first time that HDAC8 inhibits FMDV replication by regulating innate immune signal transduction and typeⅠinterferon production.To counteract the HDAC8 effect,FMDV utilizes an autophagy system to promote HDAC8 degradation.Further data showed that FMDV structural protein VP3 promotes autophagy during virus infection and interacts with and degrades HDAC8 in an AKT-m TOR-ATG5 dependent autophagy pathway.Our data demonstrated that FMDV evolved a strategy to counteract host antiviral activity by autophagic degradation of a protein that regulates innate immune response during virus infection,it provides a new reference for the pathogenesis and prevention and control FMDV.