Study On The Mechanism Of Entreovirus 71 Regulating The Biogenesis Of Small Extracellular Vesicles

Background:Small extracellular vesicles(sEVs)are a type of vesicles(EVs)surrounded by lipid membranes secreted by cells.Cell-derived sEVs are capable of encapsulating bioactive components that can induce and trigger numerous cellular response signals.Enterovirus 71(EV71)is a member of the enterovirus genus of the picornavirus family,and has a single-stranded positive RNA genome.Studies have shown that EV71-infected cells secret sEVs that contain viral nucleic acids and can establish proliferative infections in recipient cells.However,there is no study regarding how EV71 viral nucleic acids are sorted into sEVs.2C protein is one of the non-structural proteins encoded by EV71 virus,which plays an important role in the virus replication in cells and the innate immunity against the host.Considering that 2C protein has membranebinding activity and RNA-binding activity,this subject intended to explore whether 2C protein participated in the biogenesis of sEVs.Objective:This study is aimed to explore the regulatory mechanism of EV71 virus nucleic acid entering sEVs and the biogenesis of sEVs.Methods:1.Construction of EV71-2C protein expression plasmid.The constructed pcaggs-2C plasmid was identified by the electrophoresis results of plasmid double digestion and sequencing blast results.The 2C protein expression plasmid was transfected into the cells by transient transfection method,and the expression of 2C protein in the cells was identified by Western blot;the expression of 2C gene in the cells was identified by qRT-PCR technology;the GFP-with green fluorescent protein gene was identified by fluorescence microscope Fluorescent expression of 2C plasmid.2.Use transient transfection to express 2C protein in 293 T cells,and infect control cells and cells expressing 2C protein with the same amount of MOI virus.Western blot detects intracellular viral protein levels,qRT-PCR technology detects intracellular and extracellular viral nucleic acid levels,fluorescence in situ hybridization further verifies the intracellular viral nucleic acid levels,and the effect of 2C protein on the distribution of intracellular and extracellular viral nucleic acids influences were analyzed.3.The 2C protein stable expression cell line and the control cell line were screened through the G418 drug primary selection combined with flow cytometry sorting technology.The same amount of MOI virus was infected with the two cell lines for 24 hours,the supernatant was collected,and sEVs were extracted and identified.The levels of viral nucleic acid in the sEVs secreted by the two cell lines were detected by qRT-PCR technology,and the particle concentration of sEVs was detected by NTA.Compare the level of viral nucleic acid in a single sEV.In order to eliminate the influence of 2C protein on virus replication,qRT-PCR technology detects the total amount of intracellular and extracellular viral nucleic acid of the two cell lines after virus infection,compares the ratio of viral nucleic acid in sEVs to the total viral nucleic acid,and explores the effect of 2C protein on the total viral nucleic acid.The influence of viral nucleic acid into sEVs.4.Detect the expression levels of the four sEVs sorting-related genes YBX1,hn RNPA2B1,ALIX and TSG101 in the control cell line and the cell line expressing2 C protein by qRT-PCR technology,and analyze the possible regulatory mechanism of 2C protein sorting virus nucleic acid entering sEVs.5.Purifying sEVs from cell lines stably expressing 2C protein and control cell lines.And BCA total protein detection,CD63 ELISA,NTA and Western blot was applied to detect the total sEVs protein secreted by the cells,the CD63 level of sEVs,the particle concentration,and the level of surface marker proteins.The effect of 2C protein on the secretion of sEVs was analysed.6.Western blot was used to detect the expression levels of some sEVs secretion regulatory proteins in control cells and cells expressing 2C protein.Cellular immunofluorescence technology was used to detect the co-localization of 2C protein and CD63,and to explore possible regulatory mechanisms for 2C protein to promote sEVs secretion.Results:1.The 2C protein expression plasmid was successfully constructed.The 293 T cells transfected with the 2C protein expression plasmid(pcaggs-2C,p GFP-2C)contained high levels of 2C protein.2.Compared with the control cells,the intracellular viral nucleic acids level and VP1 protein level are decreased in the cells expressing 2C protein,but the extracellular viral nucleic acids level and the proportion of extracellular total nucleic acids level are increased in the cells expressing 2C protein.3.Stable expression cell line and control cell line of 2C protein are successfully constructed.After the virus infected two cell strains,compared with the control cell strain,the single sEV secreted by the cell expressing 2C protein is packaged with more viral nucleic acids;Moreover,the proportion of viral nucleic acids to the total viral nucleic acids in the sEVs expressing 2C protein is increased.4.The expression levels of the four sEVs sorting-related genes,YBX1,hn RNPA2B1,ALIX and TSG101,did not change significantly in control cell lines and cell lines expressing 2C protein.5.Compared with the control cells,the total protein concentration and concentration of sEVs,the abundance of sEVs and the expression of surface marker proteins CD63 and HSP70 of sEVs in the cells expressing 2C protein are increased,suggesting that the expression of 2C protein promotes the secretion of sEVs.6.Compared with control cells,intracellular CD63,CD9,and Rab27 A protein levels of cells expressing 2C protein are increased,and cellular immunofluorescence results shows that 2C protein could be co-located with CD63,suggesting that the mechanism of 2C promoting sEVs secretion might be mediated by CD63 which is an important molecule in sEVs biogenesis.Conclusion:EV71 promotes the sorting of viral nucleic acids into sEVs and increases the secretion of sEVs by 2C protein.