Research On The Mechanism Of Enterovirus 71 2C Protein Sorting Virus Nucleic Acid Into Exosomes

Extracellular Vesicles（EVs）are Vesicles with double membrane structure and are secreted by almost all cells.It mainly includes late endosomal derived exosomes with diameters of 50-150 nm,and Micro Vesicles（MVs）with diameters of 100-2000nm and plasma membrane budding.Enterovirus 71（EV71）,a member of enterovirus family,is one of the most important pathogens causing hand,foot and mouth disease（HFMD）.exosomes secreted by EV71 infected cells carry viral RNA and play an important role in the pathogenesis of infection,but how the EV71 RNA is specifically sorted into exosomes has not been studied yet.Considering its non-structural protein2C is RNA-binding.This study aims to explore the mechanism of EV71 non-structural protein 2C in sorting viral nucleic acids.Objective:Analysis of virus components in EV71 infected cells derived exosomes,and to explore the effect and mechanism of non-structural protein 2C of EV71 on viral nucleic acid entering exosomes.Methods:1.Use Opti Prep ^TMdensity gradient centrifugation to isolate exosomes of supernatant from EV71 infected Vero cells（Exo-EV71）.Western Blot（WB）was used to detect exosomes characteristic proteins and EV71 capsid proteins in samples of different density layers,and Nanopaticle Tracking Analysis（NTA）was used to detect particle sizes of samples with different density layers,and real-time quantitative PCR（q RT-PCR）was used to detect EV71 RNA copy number in samples with different density layers.Fluorescence in situ hybridization（FISH）was used to detect the binding between intracellular EV71 RNA to early endosomal markers.The expression levels of positive and negative chains of the virus in exosomes were detected by QRT-PCR,and the cell Cytopathic effect（CPE）was observed under ordinary microscope after co-culture of Exo-EV71 with Vero cells.2.Vero cells were transfected with 2C plasmids（hereafter called 2C-Vero）and control plasmids（hereafter called NC-Vero）using Lipo3000.Cells were infected with EV71virus in equal quantities,and exosomes and microvesicles（MVs）derived from the above two kinds of cells were extracted.q RT-PCR was used to detect the copy number of viral RNA in cells,exosomes and MVs after EV71 infection,and the proportion of the copy number of viral RNA inexosomes extracted from 2C-VERO and NC-Vero after EV71 infection to the total copy number of viral RNA（the sum of the copy number of cells,MVs and exosomes）was compared.FISH was used to test the EV71 v RNA content in the endosomes in NC-Vero cell and 2C-Vero,and the percentage of the v RNA in endosomes of the two groups of cells is compared by manders’Colocalization Coefficients（MCC）,and the effect of 2C protein on the entry of viral RNA into exosomes was analyzed.3.The expression of HSPA8 protein in Exo-EV71 was detected by WB,the binding of2C protein to HSPA8 protein was detected by Co-immunoprecipitation（CO-IP）,and the intracellular co-localization of 2C protein and HSPA8 protein was observed by laser confocal microscopy.4.Inhibition of intracellular HSPA8 protein expression with inhibitors,or construction of HSPA8 knockout strain with CRISPR technology,transfection of HSPA8 deficient cells with 2C plasmid and control plasmid,infection with EV71 virus in equal amount,extraction of exosomes and MVs from the above two kinds of cells.q RT-PCR was used to detect the copy number of viral RNA in EV71 infected cells,exosomes and MVs,and compare the proportion of exosomal viral RNA to total viral RNA.Comprehensively analyzing the role of HSPA8 protein in the process of 2C protein-mediated viral RNA entering exosomes.5.Use the same amount MOI of EV71 virus oinfected 2C-Vero and NC-Vero cells,and the cells were collected for RNA immunoprecipitation of HSPA8 protein and EV71 RNA,and exosomes from 2C-Vero were extracted by PEG precipitation method.RNA immunoprecipitation of HSPA8 protein and EV71 virus RNA in exosomes was also performed.6.Biogenic analysis was performed on the binding sites of HSPA8 protein and 2C protein using ZDOCK,a protein structure docking tool.According to the analysis results,a series of expression plasmids of truncated 2C protein were constructed.The plasmids of different fragments were transfected into Vero cells,and the possible binding sites of HSPA8 protein and 2C protein were screened by CO-IP technology.Results:1.Vero cells could secrete exosomes containing viral RNA after being infected with EV71,and the presence of viral structural protein VP1 was not detected in Exo-EV71.q RT-PCR results showed that there was a large amount of positive-strand RNA in exosomes.After EV71,Exo-EV71 and Vero cells were co-cultured with uninfected Vero cells,the cytopaic effect（CPE）appeared in the cells about 72h.2.Compared with NC-Vero infected with EV71,the proportion of viral RNA in total viral RNA in exosomes from 2C-Vero cells was significantly higher,and the difference was statistically significant（**,P<0.01）.Compared with NC-Vero cells infected with EV71,2C-Vero cells infected with EV71 increased the proportion of EV71 virus RNA in intracellular bodies,and the difference was statistically significant（*,P<0.05）.3.HSPA8 protein could directly bind to 2C protein and co-locate with 2C in cytoplasm.4.RNA immunoprecipitation results showed that HSPA8 protein could bind to viral RNA in cells.Compared with NC-Vero cells,HSPA8 protein in 2C-Vero cells could bind more viral RNA,and the difference was statistically significant（*,P<0.05）.HSPA8 protein also binds with viral RNA in exosomes.5.There was no statistical significance in the proportion of viral RNA in 2C-VERO-derived exosomes after EV71 infection compared with the NC-Vero group after inhibition or knockout of intracellular HSPA8 protein,（ns,P>0.05）.6.When the aa 305-312 region of 2C protein was deleted,no binding between 2C protein and HSPA8 was detected by immunoprecipitation.Conclusion:1.Vero cells infected with EV71 could secrete exosomes containing viral RNA,and were infectious.2.The non-structural protein 2C of EV71 can promote the entry of viral RNA into exosomes and enable more viral RNA to be transferred to recipient cells through exosomes.3.HSPA8 can bind to 2C protein,which may be a helper protein to promote viral RNA sorting.4.The binding site of HSPA8 protein and 2C protein was in aa 305-312 region.