Study On The Role Of Exosomes In Dengue Virus Type ? Infection Of THP-1 Cells

Objective:Exosomes are extracellular vesicles released after the fusion of multivesicular bodies(MVBS)with the plasma membrane of the cell.They originate from intraluminal vesicles(ILVS)during the formation of MVB.Exosomes have been shown to contain functional proteins,lipids and RNA,which mediate cell-to-cell communication and thus play a role in the physiology of healthy and diseased organisms.According to reports,dengue virus has a variety of cellular functions during infection,such as exosome-mediated cell communication.This function is regulated by delivering extracellular vectors carrying viruses or antiviral components to host cells.Therefore,studying the characteristics and functions of exosomes in viral infections will help to understand their role in virus-host interactions and provide new targets for antiviral therapy.Methods:The current methods for the isolation and purification of exosomes were evaluated,including ultracentrifugation,kit method,ultrafiltration concentration combined with kit method,iodixanol density gradient centrifugation and immunomagnetic bead affinity method.The exosomes were characterized by transmission electron microscopy,Western blot and NTA,and the quality of the extracted exosomes was compared.DENV-3 was infected with THP-1 cells,and the changes of exosome release after virus infection were detected by NTA and Western blot.RT-PCR,Western bolt,indirect immunofluorescence,real-time fluorescent quantitative PCR and other techniques were used to analyze the characteristics and effects of exosomes released by DENV-3-infected THP-1 host cells.To verify whether it can encapsulate viral nucleic acid and protein to establish proliferative infection on BHK-21 and C6/36 cells and escape the influence of neutralizing antibodies.After inhibiting the secretion of exosome by cells with GW4869 exosome inhibitor,the change of viral copy number in cell supernatant was detected to analyze the role of exosome in viral infection.Through analyzing the expression profile of exosome miRNA in the supernatant of DENV-3 infected with THP-1 by high-throughput sequencing,the differentially expressed miRNAs were screened out,and the significantly differentially expressed miRNAs were verified by real-time quantitative PCR.Results:Exosomes can be extracted by the three methods,and circular or elliptical vesicles with a diameter of 30-150 nm and a disc-like and cup-like structure can be observed under the electron microscope.Compared with the concentrated binding kit,the exosomes extracted by ultracentrifugation have clear background and less impurities.Western blot can detect the expression of exosomal marker proteins Alix,CD63 and CD81.NTA detection found that the particle size distribution of exosomes extracted by the three methods had no significant difference,and they were all single peaks.Compared with the ultracentrifugation method,the ultrafiltration combined kit method has higher exosomes concentration and total protein content,and the operation steps are simple and time-consuming;DENV infection can cause an increase in the release of host cell exosomes,further study It was found that the whole genome of DENV-3 and viral E protein exist in exosomes.At the same time,the exosomal structure can withstand RNaseA hydrolysis and 4G2 antibody neutralization,suggesting that viral nucleic acid and E protein exist in the internal cavity of exosomes.In addition,the exosomes released by DENV-3 infected THP-1 cells can mediate the infection of C6/36 and BHK-21 cells and resist the neutralization reaction.Subsequently,after using the exosome inhibitor GW4869 to inhibit the release of cellular exosomes,it was found that the viral load of DENV in the cell supernatant also decreased,indicating that the exosomes may be involved in the life cycle of DENV;After further screening and identification of differential miRNAs in exosomes released from host cells,107 miRNAs were found to be significantly enriched.Subsequently,the GO and KEGG enrichment analysis of the target genes of differential miRNAs showed that the target genes of these miRNAs were mainly enriched in the host immune/inflammatory response-related signal transduction pathways,including Pathways in cancer,Ras signaling pathway,and Rapl signaling pathway.,PI3K-Akt signaling pathway and other signal pathways,using real-time fluorescent quantitative PCR method to verify the significantly differentially expressed 6 miRNAs,which are consistent with the sequencing results.Conclusion:Ultra-high-speed centrifugation,kit method,and ultrafiltration combined kit method can extract exosomes from the cell supernatant.Iodixanol density gradient centrifugation and immunomagnetic bead method can isolate exosomes from DENV-infected cells The supernatant is purified without virus particle contamination;exosomes carrying dengue virus components can enter target cells to establish effective infection,and the addition of the exosome inhibitor GW4869 can inhibit the spread of dengue virus in cells;DENV infection changes the type and content of host miRNA in exosomes secreted by THP-1 cells.