The Effects And Underlying Mechanism Of Exosomes In The Process Of Peste Des Petits Ruminants Virus Transmission

Peste des petits ruminants?PPR?is an acute,severe and highly contagious infectious disease caused by Peste des Petits ruminants virus?PPRV?.PPR was susceptible for small ruminants such as sheep and goats,and is characterized by stomatitis,pneumonia and fever,which seriously hinders the development of sheep breeding.Exosomes are a class of nanoscale vesicles that mediate cell-to-cell communication.The biological activities of cells that uptake these exosomes can be regulated by the proteins,nucleic acids,lipids,and other substances they carry.Recent studies have shown that exosome-mediated virus escape or suppression of the host's immune system may be a key mechanism for viral pathogenicity and viral transmission.Our previous studies have shown that PPRV infection caused an increased secretion of exosomes,which implied the role of exosomes in PPRV transmission.This study focus on the role and underlying mechanism of PPRV-induced exosome secretion in viral transmission.The research contents and results are as follows:1. Vero cells and goat PBMCs was infected with attenuated strain of PPRV vaccine virus?N75/1 strain?,and the supernatant was collected.After removing dead cells and foreign proteins by low-speed centrifugation,combined with filtration technology to remove free virus particles.The exosomes were precipitated by ultra-high-speed centrifugation,and finally exosomes were enriched and purified by exosome extraction kit.The purity and concentration of the extracted exosomes were identified by nanoparticle tracking analysis?NTA?,transmission electron microscopy,and Western blot techniques.NTA results showed that the extracted vesicles peaked at about 100 nm,which was consistent with the size of exosomes.The typical sapodilla-like vesicle shape was observed under transmission electron microscope.At the same time,NTA,flow cytometry and Western blot test results showed that PPRV infection can significantly induce the increase of the above-mentioned exosome secretion levels.2. The exosomes were extracted from the supernatant of PPRV infected Vero cells,which were incubated with the recipient cells?normal Vero cells?for 72 h to detect the level of virus proliferation in the recipient cells.The results of laser confocal detection showed that a large number of PPRV-positive cells were observed in the control cells infected with PPRV and the recipient cells incubated with the exosome derived from PPRV-infected cells.Western blot analysis showed that the expression level of PPRV-N protein in recipient cells was slightly lower than that of PPRV-infected control cells,but the expression level was not significantly different.The TCID₅₀ test results showed that there was no significant difference between the virus titers in the recipient cells and the PPRV infection control cells.Further pretreated cells with different concentrations of GW4869?exosome inhibitor?for 24 h,then inoculated with PPRV,and after 48 h,the exosomes were extracted and incubated with recipient cells?normal cells?for 72 h.Western blot and TCID₅₀ test results showed the virus peplication level and virus titer in recipient cells decreased along with the increase of GW4869 concentration.The above results indicate that PPRV-infected cell-derived exosomes can establish a proliferative infection in uninfected cells.3. In view of the interaction between viral infection-induced autophagy and exosome secretion,this study further examined the effect of PPRV-induced autophagy on the level of exosome secretion.After treating cells with the autophagy inducer Rapamycin,the levels of autophagy and exosome secretion were tested.Western blot detection results showed that:Rapamycin can significantly induce the expression level of autophagy marker LC3-?in cells and the expression level of exosome marker CD63 in culture supernatant compared with the control group.The result is clear.Further,the exosomes extracted from the supernatant of PPRV-infected and rapamycin-treated cells were co-cultured with recipient cells?normal cells?to detect the PPRV replication level in the recipient cells.The results showed that the PPRV-infected and rapamycin-treated group enhanced PPRV replication levels in recipient cells compared to the PPRV-infected control group.To further analyze the effect of autophagy induced by PPRV infection on exosome secretion,sh RNA that interfered with autophagy-related molecules?ATG7 and Beclin-1?were transfected into cells and then inoculated with PPRV to detect the level of exosome secretion.The results showed that the inhibition of autophagy by interfering with ATG7 significantly inhibited the level of exosome secretion,and at the same time suppressed the level of virus release dependent on the exosome pathway.4. TSG101 plays an important role in regulating the content of exosome loading.In order to determine the effect of TSG101 on the regulation of exosome-dependent viral release pathways during PPRV infection,PPRV was first infected with Vero cells to detect the expression of TSG101 protein and exosome secretion level in the cells.Western blot and laser confocal detection results showed that:PPRV infection can enhance the expression of TSG101protein in cells.Subsequently,cells were transfected with si RNA that interfered with TSG101or plasmids containing TSG101 gene,and then inoculated with PPRV to detect the level of exosome secretion and the level of PPRV nucleic acid in exosomes.The results showed that interfering with TSG101 significantly reduced the level of PPRV nucleic acid in exosomes,while overexpressing TSG101 increased the level of PPRV nucleic acid in exosomes.In addition,PPRV replication levels in cells co-cultured with knockdown TSG101 cell-derived exosomes were significantly lower than in the control group.Compared with the transfection control group,knockdown or overexpression of TSG101 had no effect on the level of exosome secretion.5. To further clarify the role of PPRV protein molecules in inducing autophagy and exosome secretion,cells were transfected with plasmids containing PPRV C,V,N,H,F,and M genes,respectively.It was found that the plasmids transfected with C-HA,H-HA and N-HA significantly induced the expression of LC3-II,while promoting the expression levels of TSG101 and CD63.After transfecting cells with plasmids expressing fluorescent virus proteins?C-GFP,N-GFP and H-GFP?,laser confocal detection detected that C protein,N protein and H protein all co-located with TSG101.And Co-IP detection showed that the C protein,H protein and N protein of the virus all interacted with TSG101.In summary,PPRV C,H,and N proteins play a key role in exosome-mediated viral cell transmission by up-regulating TSG101 and inducing autophagy.In summary,PPRV-infected cell-derived exosomes can carry infectious PPRV nucleic acids to establish proliferative infections in recipient cells.PPRV C,H,and N protein play a key role in selective loading PPRV infectious nucleic acid into exosomes and promote exosomal release.This study reveals the important role of exosomes in the transmission of PPRV,and the role played by endogenous TSG101 and autophagy in regulating exosomal loads and secretion release,which providing new ideas for the prevention and control of Peste des petits ruminants.