The Mechanism Of Enterovirus 71 Non-structural 3A Protein On Exosomes Secretion

Enterovirus 71(EV71),a member of picornaviruses family,is one of the most important pathogens that cause severe hand,foot and mouth disease(HFMD),and is considered to be the most crucial neurotropic enterovirus after poliovirus.EV71 is often prevalent in the Asia-pacific region,causing major public health problems.Exploring the pathogenesis of EV71 and seeking a new antiviral target remains an important task.Exosomes are extracellular vesicles secreted by a variety of cells with a diameter of 50-150nm.They mediate the cell-to-cell communication by transferring functional proteins,lipids and RNA.Studies have shown that exosomes derived from virus-infected cells containing viral components can amplify viral spread and infection.3 A protein,one of the non-structural proteins of EV71,plays a critical role in the process of virus infection and replication as well as in mediating the interaction between virus and host.However,the role of 3A protein in exosomes secretion is still unknown.Objective:This study is to analyse the role of exosomes derived from EV71 infected cells in viral transmission;to investigate the regulatory effect of EV71 infection on exosomes secretion in vivo and vitro and elucidate the role of EV71 non-structural protein 3A on exosomes secretion,searching for the molecular mechanism of 3A regulating exosomes secretion in vivo and vitro;to provide a novel research point for the study of the pathogenesis of EV71.Methods:1.Exosomes derived from EV71 infection possess infectivity.Exosomes(Exo-EV71)from cells infected with EV71 were preparated by differential centrifugation combined with immune affinity magnetic beads,and quantified by BCA microprotein detection kit(BCA assay).The morphology and size of Exo-EV71 were observed by transmission electron microscope(TEM).The expression of Exo-EV71 surface marker proteins was detected by western blot.The particle size and concentration distribution of Exo-EV71 were analyzed by nanoparticle tracing technique(NTA).After Exo-EV71 treatment,the CPE of SK-N-SH cells and Hela cells were observed by electron microscopy.The expression of viral VP1 protein in cells was detected by western blot.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the levels of viral RNA in cells.2.Effect of EV71 infection on the exosomes secretionExosomes(Exo-EV71)and exosomes(Exo-mock)from EV71 and mockinfected cells were preparated by differential centrifugation combined with immunoaffinity and magnetic beads.The number of exosomes released from Exo-EV71 and Exo-mock groups was compared by BCA assay,Exosomal CD63-ELISA(ELISA),western blot and NTA.The model of EV71 infected one-day-old ICR mice was constructed,and exosomes from tissues of mice were prepared by differential centrifugation combined with sucrose density gradient centrifugation.The number of exosomes from tissues in the infected and mock-infected mice was compared by BCA assay and ELISA.3.Effects of non-structural protein 3A on exosomes releasePCDNA3.1-3A recombinant plasmid was used to construct cell line with stable expression of 3A protein.Western blot and cell immunofluorescence techniques(IF)were used to identify the stable expression of 3A protein.BCA assay,western blot and NTA were used to compare the number of exosomes released by cell line with stable expression of 3A protein with those that were not loaded.The morphology and quantity of intracellular vesicles(MVBs)and intracavitary vesicles(ILVs)were compared by TEM.The expression of intracellular CD63,EEA1 and LC3B was compared by western blot and IF.The 3A mutant strain was constructed.The 3A mutant strain with different amino acids mutations and wild strain were used to infect cells,exosomes in the supernatant of the infected cells were isolated,and the number of exosomes was compared by BCA assay,ELISA,western blot and NTA.The 3A-P18R mutant strain and the wild strain were used to infect ICR mice.Differential centrifugation combined with sucrose density gradient centrifugation was used to isolate exosomes from the mice tissue.The number of exosomes from tissues in the 3A-P18R mutant strain and the wild strain infected mice was compared by the BCA assay,ELISA and NTA.The morphology and quantity of MVBs and ILVs in brain tissue were examined by TEM.4.Non-structural protein 3A enhanced exosomes secretion through reducing the degradation of Rab-27aProteins interacting with 3A were screened using the protein interaction network.Intracellular staining was performed in the cell line with stable expression of 3A protein,and the cellular localization of 3A and Rab-27a was detected by cell IF.The combination of 3A and Rab-27a protein was identified by immunoprecipitation(IP).Small interfering RNA(si-RNA)was used to knockdown the expression of intracrllular Rab-27a protein in cell line with stable expression of 3A protein,qRT-PCR and western blot were used to verify the interference efficiency of Rab-27a.BCA assay and ELISA were used to compare change of exosomes number from the cell line with stable expression of 3A protein after inhibition of Rab-27a.Western blot and qRT-PCR were used to detect the protein expression and mRNA levels of Rab-27a in the cell line with stable expression of 3A protein and control cells to observe the effect of 3A protein on Rab-27a expression.Protein synthesis inhibitor,proteasome inhibitor and lysosome inhibitor were used to treat cell line with stable expression of 3A protein,the protein expression of Rab-27a was detected by western blot.Ubiquitination of Rab-27a protein was detected by IP and western blot.The 3A mutant strain and the wild strain were used to infect the mice.QRT-PCR was used to detect the mRNA levels of Rab-27a in tissues.The protein expression of Rab-27a in different tissues was compared by western blot.The intracellular expression of Rab-27a protein in different tissues was compared by IF.Results:1.EV71-infected cells can secret exosomes;Exo-EV71 showed a typical discoid vesicle structure,expressing exosomal surface marker proteins CD63,TSG101 and HSP70 without expressing the cell endoplasmic reticulum marker protein Calnexin.Exosomes had uniform particle size distribution and were approximately 100-120nm in diameter.Exo-EV71 is infectious and can replicate in the receptor cells to cause significant CPE.Exo-EV71 resisted the neutralizing antibody from HFMD patients.2.In vitro,the total protein,particle abundance and surface marker proteins expression of exosomes from EV71-infected cells were significantly higher than that from mock-infected cells.In vivo,the exosomal total protein and particle abundance from brain and intestinal tissues in EV71-infected mice increased significantly compared with those from mock-infected mice.3.Compared with control cell line,the exosomal total protein,particle abundance,particle concentration and expression of surface marker proteins from cell line with stable expression of 3A protein were significantly increased.The number of intracellular MVBs and ILVs in cell line with stable expression of 3A decreased without changing size.The expression of intracellular CD63 was decreased,but there was no difference about EEA1 and LC3B.Compared with the wild strain,the total protein,particle abundance,particle concentration and expression of surface marker proteins of exosomes released from 3A mutant strain infected cells were all decreased,but the particle size of exosomes was unchanged.Compared with the wild strain infected mice,the total protein,particle abundance and particle concentration of exosomes from brain and intestinal tissues of the mice infected with the 3A mutant strain were decreased,while the number of exosomes from lung and cardiac tissues remained unchanged.The morphology and size of MVBs and ILVs in brain tissue of mice infected with the 3 A mutant strain remained unchanged,while the number of ILVs in brain tissue was decreased.4.Rab-27a is the target protein of 3A through the protein interaction network.3A protein interacting with Rab-27a protein was verified through IP,3A protein and Rab-27a protein co-located in the cytoplasm.The release of exosomes from the cell line with stable expression of 3A was decreased after inhibiting Rab-27a.Compared with control cell line,Rab-27a protein expression in cell line with stable expression of 3A protein was increased,and the mRNA level was unchanged.3A protein stabilized the protein level of Rab-27a by reducing the ubiquitination level of Rab-27a.Compared with the group of wild strain infected mice,the expression of Rab-27a protein in brain tissue from the 3A mutant strain infected mice was decreased,and the mRNA level remained unchanged.Conclusion:The exosomes secreted from EV71-infected cells contain viral components and can establish proliferative infections.EV71 infection promotes exosomes secretion,which is mediated by the non-structural protein 3A of EV71.3A protein interacting with Rab-27a prevents the degradation of Rab-27a by the protease-ubiquitination pathway,increasing the protein level of intracellular Rab-27a which promotes the docking of MVBs with plasma membrane to increase exosomes secretion.The increased secretion of infectious exosomes is a crucial factor to promote the further transmission and infection of EV71.