Proteomics-based Analysis Of EV-A71-mediated Neuronal Response And NR1H3 Regulation Of Viral Replication And Its Mechanis

Hand,foot,and mouth disease(HFMD)is a common infectious disease caused by multiple types of enteroviruses,with several outbreaks occurring worldwide,especially in Asian countries.From 2008 to 2019,the incidence rate of HFMD in China ranked first among category C infectious diseases and has become one of the most important category C infectious diseases,significantly affecting the health of infants and young children.There are more than 20 kinds of pathogens that can cause HFMD,among which EV-A71 is the main pathogen causing severe or fatal HFMD.The unclear pathogenic mechanism of EV-A71 virus greatly hinders the development of antiviral drugs.Currently,systematic research on the pathogenic mechanism of EV-A71 virus infection is still lacking,particularly the panoramic analysis of the entire process of EV-A71 virus invasion into the nervous system.Therefore,it is urgent to comprehensively and systematically explore the mechanism of EV-A71 virus invasion into the nervous system and need for development of novel antivirl therapies.Protein phosphorylation and dephosphorylation modifications are one of the most common post-translational modifications that regulate all life activities,including cell proliferation and differentiation,signal transduction,apoptosis,neural activity,muscle contraction,and tumor occurrence.Phosphorylation of viral proteins mainly regulates virus genome replication and transcription,targets protein activity and subcellular localization,assists virus particle assembly and release,and affects virus infectivity and virulence.At the same time,phosphorylation of host proteins can affect protein activity activation,cell localization,and binding to receptors,ligands,or downstream signaling molecules,thereby regulating the cell cycle,cell survival,signal transduction,and host antiviral innate immunity or immune response.Proteomics technology has been widely used in the study of virus-host interactions.By analyzing the host-virus interaction through proteomics and phosphoproteomics,it can help to understand the pathogenesis of virus infection more deeply.We utilized a human neuroblastoma cell line SK-N-SH model infected with Enterovirus A71 to quantitatively analyze the comprehensive systemic dynamic protein levels and phosphorylation modification changes of host cell proteins and viral proteins using label-free quantitative proteomics and phosphoproteomics methods at different time points of control,12 and 24 hours post of infection.Quantitative proteomic analysis found that EV-A71 infection regulated host cell pathways such as autophagy,neurodegeneration,and antiviral immune response,remodeled important cellular metabolic processes such as translation,cytoskeleton,glycolysis,and nucleic acid metabolism.Up-regulated differentially expressed proteins such as ICAM1,PTEN,and down-regulated differentially expressed proteins such as MAPK14 may play important roles in the process of EV-A71 virus infection.Quantitative phosphoproteomic analysis found that both host and viral proteins had phosphorylation modifications.Differentially phosphorylated host proteins mainly participated in signal pathways such as virus replication,endocytosis,cytoskeleton regulation,RNA splicing,and axon guidance.Upregulated differentially phosphorylated proteins such as EGFR,SMARCA1 and downregulated differentially phosphorylated proteins such as HDAC,MYC,PTK2 may play important roles in the interaction between EV-A71 virus and host.Kinase-substrate prediction was based on differentially phosphorylated proteins and their modification sites,and kinase activity was predicted through the quantity and abundance of substrates.The phosphorylation kinase activities of ATR,CDK2,CDK7,AKT,PRKACB,MAP2K7 and other kinases were upregulated in the 12 h and 24 h virus-infected groups.Moreover,up-regulated phosphorylation kinases such as CK1a,CDK2,CLK1,DAPK3,NEK2,IKK could target multiple phosphorylated protein substrates.The RT-PCR experiment showed that ATR inhibitor Ceralasertib,CDK2 inhibitor Bosutinib,SGK1 inhibitor Flavin mononucleotide,JNK2 inhibitor Minocycline,MAP2K7 inhibitor Pimasertib,and IKKβ inhibitor Acetylcysteine could significantly inhibit EV-A71 virus replication,suggesting that CDK2 and IKK kinases play a critical role in the interaction.Furthermore,We discussed the identification of phosphorylation modification sites on various EV-A71 virus proteins,including VP1,VP4,2A,2B,2C,3A,and 3D.Specifically,We identified S184 and T356 as important phosphorylation sites on the highly conserved 3D protein.We used EV-A71 replication systems to confirm that a mutation at the S184 site completely attenuated virus genome replication,and a mutation at the T356 site significantly attenuated virus genome replication.Additionally,We used the FY-08 recombinant infectious clone system to show that mutations at these sites also affected virus production and infectivity.Through a combination of proteomics and phosphoproteomics analyses,We found that differentially expressed proteins and phosphorylated proteins were mainly enriched in signaling pathways such as AMPK,axon guidance,and autophagy pathways,which regulate biological processes such as cell autophagy,growth and protein synthesis,cell survival and cytoskeletal assembly,and axon guidance and transport.Furthermore,Western blot experiments showed that EV-A71 infection triggered the mTORCl signaling pathway.In addition,we investigated the effect of NR1H3 on EV-A71 virus replication and its mechanism.The results showed that knocking down NR1H3 could inhibit the replication level of EV-A71 virus,and knocking out NR1H3 could also inhibit the replication of EV-A71 virus.Restoring NR1H3 could rescue EV-A71 virus replication levels.NR1H3 is an important host factor on which EV-A71 virus replication depends.In addition,through RT-PCR and immunofluorescence,we found that knocking out NR1H3 could inhibit the early stages of EVA71 virus adsorption and invasion.Mechanistically,NR1H3 could inhibit the production of type Ⅰ interferon.Knocking out NR1H3 upregulated the production of type Ⅰ IFN and activated downstream antiviral ISGs expression such as APOBEC3,IFIT1,OAS,MX1,MX2,IFI,IFITM1,and TRIM22,directly inhibiting viral invasion,viral genome replication and translation,and other aspects of viral activity.At the same time,knocking out NR1H3 lead to a decrease in total cellular cholesterol,and cholesterol treatment can increase virus levels.This may affect the expression levels and distribution of cell membrane surfaces,thereby inhibiting the early stages of EV-A71 virus.In summary,we quantitatively analyzed comprehensive systemic dynamic phosphoryl modification changes in host cell proteins and viral proteins at different time points in control,12 and 24h post of infection by Label-free quantitative proteome and phosphoproteomics,using the SK-N-SH model of human neuroblastoma cells infected by enterovirus EV-A71.We cleared that EV-A71 virus could remodel cell biological processes such as translation,splicing and nucleic acid metabolism in nervous cells and autophagy with mTOR signal transduction pathway,identifing inhibitors targeting activated phosphokinase capable of inhibiting EV-A71 viral replication.The EV-A71 3D S184 site and the T356 site were also found as key sites for EV-A71 virus genome replication.We also explored NR1H3 as a host factor essential for EVA71 virus replication,affecting the early adsorption and invasion stages of the EV-A71 virus probably through type Ⅰ IFN signaling and regulating the expression level and distribution of the cell membrane surface.In this study,we further systematically explored the signal transduction of the virus of enterovirus EV-A71 infection in the nervous system and the antiviral response mechanism of the host,thus revealing the molecular mechanism of EV-A71 virus invasion into the nervous system and the potential drug target of antiviral therapy.