Research On The Analysis Of Protein Composition And Functions Of SEVs Secreted By EV71-infected Cells

Enterovirus 71（EV71）belongs to group A of the genusEnterovirus within family Picornaviridae.It is a nonenveloped single-stranded positive strand RNA virus that mainly causes hand,foot and mouth disease（HFMD）and herpetic angina.In severe cases,EV71infects the central nervous system and causes nervous system diseases,such as aseptic meningitis,brainstem encephalitis and cerebellar encephalitis.Small extracellular vesicles（sEVs）are lipid bilayer encapsulated vesicles with a diameter of less than 200nm,which can carry a variety of bioactive substances such as proteins,nucleic acids and lipids to participate in various biological activities between cells and play a role in cell-to-cell communication.Human rhabdomyosarcoma cells（RD）are susceptible cells to EV71 virus.Our previous study showed that sEVs secreted by EV71-infected RD cells（sEVs-EV71）can infect recipient cells with viral components.But how sEVs-EV71 releases contents in recipient cells to infect recipient cells has not been reported.The aim of this study is to screen the differential protein profiles of sEVs-EV71 and sEVs-mock by 4D-lable-free proteomics technology,and then to conduct relevant functional studies on the proteins with significant differences to explore the protein composition and related functions of sEVs derived from EV71 infected cells.Objective:1.To screen and analyse the differential proteome of sEVs secreted by EV71 uninfected cells and infected cells2.To explore the function of differential protein LRBA in the process of sEVs-EV71infecting recipient cell and its regulation mechanismsMethods:1.Extraction and identification of sEVs from EV71-infected and uninfected RD cellssEVs secreted by EV71 infected and uninfected RD cells（sEVs-EV71/sEVs-mock）were isolated and purified by differential centrifugation and size exclusion chromatography columns respectively.Nanopaticle Tracking Analysis（NTA）was used to detect the particle size.Transmission Eletron Microscope（TEM）was used to observe the morphological structures of sEVs and Western Blot（WB）was used to detect characteristic protein of sEVs.Real-time quantitative fluorescent PCR（q TR-PCR）was used to detect levels of viral nucleic acid and WB was used to detect the EV71 structural protein VP1 of sEVs-EV71.sEVs-EV71 were co-cultured with recipient cells,and ordinary optical microscope was used to observe the cell cytopathic effect（CPE）in recipient cells.2.Analysis and screening of the differential protein profiles of sEVs-mock and sEVs-EV714D-label-free quantitative proteomics technology was used to detect the proteomic compositions of sEVs-EV71 and sEVs-mock and the differences in protein expression between sEVs-mock and sEVs-EV71.Domain prediction software Interproscan was used for domain prediction and functional analysis of differential proteins.The LPS-responsive,beige like anchor protein（LRBA）,which may affect the function of sEVs-EV71,was screened according to the degree of protein difference and protein function retrieval.3.Analysis of the effects of LRBA on sEVs-EV71 infected recipient cellsCRISPR/Cas system genome editing technique was used to construct the LRBA knockout African green monkey kidney cell lines and LRBA gene wild type cell lines（Vero LRBA KO and Vero LRBA WT）.sEVs-EV71 secreted by Vero ^{LRBA KO}and Vero ^{LRBA WT}at 24 hours after EV71（MOI=1）infection(sEVs-EV71^{LRBA WT}and sEVs-EV71^{LRBA KO})were extracted respectively.sEVs-EV71^{LRBA WT}and sEVs-EV71^{LRBA KO}containing the same amount of EV71 RNA were co-cultured with recipient cells for 24 hours.The EV71 RNA level in the recipient cells was detected by q RT-PCR and the viral structural protein VP1 level in the recipient cells was detected by WB to analyze the role of LRBA in sEVs-EV71 infected cells.4.Analysis of the effect of LRBA on sEVs-EV71 transferring virus nucleic acid to recipient cellsDio-labeled sEVs ^{LRBA WT}and sEVs ^{LRBA KO}were incubated with recipient cells on ice for1h,and the binding of sEVs to the surface of recipient cells was observed by fluorescence microscopy.The recipient cells were incubated at 37℃for 1h,and the internalization of sEVs in the two groups was observed by fluorescence microscopy to analyze the effect of LRBA on the endocytosis of sEVs by recipient cells.The recipient cells were infected with sEVs-EV71^{LRBA WT}and sEVs-EV71^{LRBA KO}containing the same amount of viral RNA.The levels of viral non-structural protein 2C in recipient cells were detected by Immunofluorescence（IF）at different time points.The effect of LRBA on the release of sEVs-EV71 nucleic acid in recipient cells was analyzed.5.Analysis of the effect of LRBA on membrane fusion of sEVs and endosomes in recipient cellssEVs ^{LRBA WT}and sEVs ^{LRBA KO}labeled by octadecyl rhodamine B chloride（R18）were added to the supernatant of reciticent cells,respectively.The fluorescence released by membrane fusion between R18-labeled sEVs（R18-sEVs）membrane and intracellular endosomal membrane was detected by IF at different time points to analyze the effect of sEVs membrane protein LRBA on membrane fusion of sEVs in recipient cells.The co-localization of R18-sEVs with early endosome（EE）,late endosome（LE）and lysosome was detected by IF to analyze the sEVs fusion sites and effects of LRBA on the location of membrane fusion.6.Explore the possible mechanism of LRBA promoting membrane fusion by forming dimer through homologous oligomerizationEarly and late endosomes and lysosomes of recipient cells were labeled with fluorescence,and the co-localization of LRBA with early and late stage bodies and lysosomes was detected by IF to analyze the subcellular localization of LRBA.R18-labeled sEVs ^{LRBA WT}was added to the supernatant of Vero ^{LRBA KO}and Vero ^{LRBA WT}recipient cells,respectively.The fluorescence released by membrane fusion of R18-labeled sEVs and endosomal membrane of recipient cells was detected by IF to analyze the effect of LRBA in recipient cells on the membrane fusion of sEVs in recipient cells.After sEVs-EV71^{LRBA WT}and sEVs-EV71^{LRBA KO}were incubated with recipient cells at37℃for 110 minutes,membrane protein was extracted to detect monomeric LRBA and oligomerization LRBA levels in recipient cells by reducing SDS-PAGE and non-reducing Native PAGE to observe the homologous oligomerization of LRBA molecules on the endosomal membrane of recipient cells and LRBA protein.Co-Immunoprecipitation（Co-IP）was used to detect the interaction between the PH domain and the BEACH domain of LRBA.Results:1.The sEVs secreted by EV71 infected cells had a bilayer vesicle structure,the size of which was concentrated around 100nm.sEVs contained the sEVs characteristic protein molecules CD63、HSP70 and TSG101,and sEVs-EV71 contained positive-sense viral RNA and could infect the recipient cells.2.The differential protein profiles of sEVs-EV71 and sEVs-mock were obtained by mass spectrometry screening.There were 42 distinct proteins that went up significantly.Among them,LRBA protein in sEVs-EV71 was involved in coupling signal transduction and vesicle trafficking,and mainly distributed in the intracellular endsome membrane system.3.The significant difference protein LRBA in sEVs-EV71 was mainly located on the membrane surface of sEVs,and the absence of LRBA membrane protein inhibits sEVs-EV71to infect recipient cells.4.The analysis of the three stages of sEVs entry into the recipient cells showed that the absence of sEVs membrane protein LRBA did not affect the binding and internalization of sEVs to the recipient cells,but affected the release of viral RNA of sEVs-EV71 in the recipient cells.5.In recipient cells,the absence of sEVs membrane protein LRBA inhibited the membrane fusion of sEVs with endosomes,resulting in a decrease in the membrane fusion ratio of sEVs with endosomes and an increase in the membrane fusion ratio of sEVs with lysosomes.6.The absence of LRBA protein in the receptor cells inhibits the membrane fusion between sEVs and the endosomes of the recipient cells,and its intracellular localization was mainly located in the endosomes.Oligomerization LRBA in recipient cells added sEVs-EV71^{LRBA WT}was significantly increased,indicating that LRBA may form dimers to promote membrane fusion through oligomerization of LRBA on the sEVs-EV71 membrane and LRBA on the endosomes of the recipient cells.Conclusion:LRBA,the membrane protein of sEVs,and LRBA on the endosomal membrane of recipient cells can oligomerization to form LRBA dimer to narrow the membrane distance,promote membrane fusion,achieve endosomal escape of sEVs-EV71,release the viral RNA in sEVs-EV71 to the cytoplasm,and enhance the infection of recipient cells with sEVs-EV71.