Exosome-mediated Mechanism Of Foot-and-mouth Disease Virus Infection

Exosomes are small membrane-enclosed vesicles that can transfer biologically active proteins,lipids,and nucleic acids.After viral infection,the exosomes secreted by the host cells carry the active components of the virus or host cells,and the exosomes are involved in regulating viral infection or host immune response.The role and mechanism of exosomes derived from foot-and-mouth disease virus?FMDV?-infected host cells in FMDV infection is unclear.In this study,exosomes isolated and purified from FMDV infected cell supernatants?FMDV-exosomes?and uninfected?MOCK-exosomes?were used to reveal FMDV-exosomes for FMDV infection.The main contents of the impact are as follows:1.Isolation,identification and composition analysis of exosome from PK-15 cells infected with FMDVIn this study,FMDV?A/GDMM/CHA/2013?strain was used to infect pig kidney cell PK-15,and the infected cell supernatant was collected.The FMDV-exosomes were isolated and purified by differential centrifugation and immunopurification.The purified product was detected by Western blotting?WB?,transmission electron microscopy?TEM?and dynamic light scattering?DLS?,and it was confirmed that the exosomes were isolated.The FMDV gene was divided into 7 fragments,and the RNA in FMDV-exosomes was extracted.RT-PCR could successfully amplify 7 fragments,which proved that FMDV-exosomes contained full-length FMDV nucleic acid sequences.The FMDV-exosomes protein expression profile was identified by liquid-phase mass spectrometry?LC-MS/MS?.A total of 1133 host proteins and 9 FMDV proteins?VP1,VP2,VP3,VP4,2B,2C,3A,3C,3D?were identified;At the same time,the PK-15-CD63-GFP cell line stably expressing CD63-GFP was used to verify the mass spectrometry results,and it was found that red fluorescence?FMDV-VP3?was surrounded by green fluorescence?exosomes?,which proved that FMDV-exosomes contained FMDV protein.2.FMDV-exosomes and FMDV infectionPK-15 cells were incubated with DiO-labeled?green?FMDV-exosomes,and green fluorescence was observed in the cells by confocal microscopy,demonstrating that the isolated FMDV-exosomes could enter the host cells.FMDV-exosomes were inoculated with PK-15 and IBRS-2 cells.FMDV-exosomes could cause cytopathic lesions in both cells,and qRT-PCR could detect viral replication,demonstrating that FMDV-exosomes could infect FMDV susceptible cells.FMDV-exosomes were inoculated into RAW264.7cells,and viral replication was detected by qRT-PCR,demonstrating that FMDV-exosomes could infect FMDV non-susceptible cells.FMDV-exosomes were treated with FMDV neutralizing antibody and incubated with PK-15 cells.FMDV-exosomes were still able to infect PK-15 cells,demonstrating that FMDV-exosomes infected cells were not affected by neutralizing antibodies.To further validate the pathogenicity of FMDV-exosomes,FMDV-exosomes were inoculated into 3day old Balb/C mice,and the death of suckling mice occurred at 48 hours,and FMDV replication was detected,confirming that FMDV components in FMDV-exosomes could replicate in vivo.3.FMDV-exosomes and FMDV replicationThe total protein content of FMDV-exosomes and MOCK-exosomes was quantified by BCA protein concentration assay.FMDV-exosomes and MOCK-exosomes were incubated with PK-15 cells,and FMDV was inoculated.It was found that the FMDV-exosomes group had lower FMDV load,which proved that FMDV-exosomes inhibited FMDV replication.Using si-Rab27a to interfere with the host protein Rab27a,it was found that the intracellular and extracellular viral load was higher than the control group 18 h after infection with 1 MOI virus;treatment of cells with 0,5,10?mol of GW4869 and inoculation of FMDV revealed that intracellular and extracellular viral load was higher than that of the control group in a dose-dependent manner.The above results confirmed that reducing the secretion of exosomes is beneficial to FMDV replication.In order to explore the signal pathways in which FMDV-exosomes inhibited viral replication,FMDV-exosomes were inoculated into PK-15,Vero,and RAW264.7 cells,it was found that FMDV-exosomes up-regulated TNF-?transcription on all three cells.4.FMDV replication and secretion of exosomesWB was used to detect exosomess marker proteins,and the BCA protein concentration assay was used to quantify the total amount of exosome protein,it was found that the exosomes isolated from the FMDV-infected group were less than the uninfected group,demonstrating that FMDV replication reduced exosomes secretion.PK-15 cells were inoculated with FMDV,and the host protein Rab27a was reduced with FMDV replication,but the transcription level was not affected.The CQ,NH₄Cl,Z-VAD-FMD,and MG132 protein degradation inhibitors were used to suggest that FMDV degradation of the Rab27a by the autophagolysosomal pathway.WB assay found that overexpression of Rab27a followed by FMDV infection,the secretion of exosomes did not decrease,demonstrating that FMDV inhibits exosomes secretion by degrading Rab27a.At the same time,overexpression of Rab27a inhibited FMDV replication,indicating that FMDV promotes FMDV replication by inhibiting exosomes secretion.Twelve FMDV encoded proteins were transfected,and FMDV-2C degraded the host protein Rab27a in a dose-dependent manner.Further studies have found that FMDV-2C could reduce the secretion of exosomes.