Research On The Function And Mechanism Of Exosomesin PRRSV Infection

Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space,and are 30-150 nm in size.They participate in intercellular communication and transfer of biologically active proteins,lipids and nucleic acids.Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins,genetic materials or even virions to mediate cell-to-cell communication and/or virus transmission.Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins,and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins.However,the role of exosomes in PRRSV infection remains unclear.Based on the above issues,we conducted a study on the role of exosomes in the spread and infection of porcine reproductive and respiratory syndrome virus.In this study,purified exosomes isolated from PRRSV-infected cells are shown to contain viral genomic RNA and partial viral proteins.Furthermore,exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and-nonsusceptible cells.More importantly,exosome-mediated infection is not completely blocked by PRRSV-specific neutralizing antibodies.The major studies include:1.Isolation and purification the exosomes derived from PRRSV-infected cells?PRRSV-exosome?Because PRRSV virions and exosomes share similar sizes and buoyant densities,the conventional ultracentrifugation method cannot efficiently separate exosomes from viral contamination.In order to obtain exosomes with greater purity,we used a stringent isolation method to extract and purify exosomes from PRRSV-infected cells,with polyethylene glycol?PEG?enrichment precipitation and ultracentrifugation combined with CD63 immunomagnetic beads affinity purification.The purified exosmes were characterized by an analysis of exosomal markers with western blotting.Three representative exosome markers,Alix,CD9 and CD63 were detected.Transmission electron microscopy also verified the presence of exosome-sized,vesicle-shaped particles of the purified exosomes.The purified exosomes were further characterized by immune-gold labeling with antibodies against Alix,the exosome marker and nsp2 to demonstrated the purification.2.PRRSV-exosome contain viral components To characterize the contents of exosomes purified from PRRSV-infected cells,a liquid chromatography-tandem mass spectrometry?LC-MS/MS?analysis was performed.Our LC-MS/MS analysis showed that 216 porcine proteins were present in the purified exosomes derived from PRRSV-infected cells.Among the proteins identified,it was revealed the presence of three viral proteins:GP5,M and N.We also used reverse transcription?RT?-PCR to investigate whether the exosomes derived from PRRSV-infected cells contain viral RNAs.Because it is difficult to amplify the complete genomic RNA by a single PCR,the whole genome of PRRSV was divided into five overlapping fragments?A,B,C,D and E?to be amplified.3.Exosomes transmit PRRSV and establish productive infections in susceptible and nonsusceptible cells The exosomes purified from PRRSV-infected cells were incubated with na?ve PK-15^CD163cells and indirect immunofluorescence assay?IFA?for viral N protein was performed at 36 hpi to confirm infection.We also tested Marc-145 cells,another cell line permissive of PRRSV,a similar productive infection was established after treated with PRRSV-positive exosomes,whereas no cytopathic effect was observed in cells treated with the control exosomes.The expressions of viral proteins N,GP4 and nsp2 could also be detected with western blotting in PK-15^CD163and Marc-145 cells.TCID₅₀0 assays were performed to determine the viral titers in the exosome-treated PK-15^CD163and Marc-145 cells,and cell-culture-derived PRRSV was used as the control.The results showed that PRRSV-positive exosomes generated high viral titers in both PK-15^CD163and Marc-145 cells,which were similar to the levels seen in cells infected with the cell-culture-derived PRRSV.Interestingly,we found that a productive infection could be established in PK-15 cells by PRRSV-positive exosomes independent of PRRSV receptor.4.Inhibition of exosome release impairs PRRSV transmission mediated by exosome we mimicked the exosome transfer from PK-15^CD163cells to PK-15 cells using a coculture model?Transwell system?in the presence of GW4869.PRRSV RNAs from the cells in both top and bottom chambers were quantified with real-time RT-PCR and western blot,the results showed that PRRSV RNA copy number was markedly lower in the PK-15 cells,at the same time,cell samples from both top and bottom chambers were collected and subjected to western blotting analysis with antibody against PRRSV nsp2.The protein expression level of each group maintained in accordance with the PRRSV RNA level.These results suggest that GW4869 has a direct effect on the transmission of PRRSV mediated by exosomes,indirectly supporting our conclusion that exosomes derived from PRRSV-infected cells can establish a productive infection in nonsusceptible cells.5.Exosome-mediated PRRSV infection is not blocked by PRRSV-specific NAbs.We also investigated whether exosome-mediated PRRSV transmission is blocked by PRRSV-specific NAbs.PRRSV-positive exosomes or free PRRSV suspensions were incubated with the purified NAbs,and then transferred into a monolayer of PK-15^CD163cells and Marc-145 cells.Cells were fixed for IFAs with mAb directed against PRRSV N protein.The number of infected cells decreased significantly in the group treated with free PRRSV and NAbs than in the group treated with PRRSV without NAbs,however,there was almost no difference between the PRRSV-exosome group?mock-treated PRRSV-positive exosomes?and the NAbs-treated PRRSV-positive exosomes.The results of western blotting used to detect the expression of PRRSV N and nsp2 proteins confirmed the IFA results.Consistent with the IFA results and western blotting,the PRRSV RNA levels were significantly reduced in both PK-15^CD163cells and Marc-145 cells treated with NAbs-treated PRRSV,but only barely reduced in cells treated with NAbs-treated PRRSV-positive exosomes.6.PRRSV N contribute to NF-?B pathway activation and expression of inflammatory cytokines through exosomal pathway Previous studies have verified that PRRSV infection activates the NF-?B pathway through I?B degradation.it is essential to understand the biological effects of target cells triggered by exosomes which can delivery PRRSV N protein to healthy cells.To this end,we transfected the HEK-293 T cells with the eukaryotic expression plasmid of PRRSV N protein and transfected the blank plasmid as a control.After transfection,the cell culture supernatant was collected and the exosomes were extracted using reagent precipitation.The exosomes marker proteins were detected by western blotting.The exosomes derived from N-transfected cells were capable of transporting PRRSV N protein to normal PK-15^CD163D163 cells.The activity of NF-?B was detected by dual luciferase assay and we found that N-exosomes activated the NF-?B pathway compared with the control group.To further explore the expression of inflammatory cytokines induced by N-exosomes,PK-15^CD163D163 cells were treated with N-exosomes and control exosomes,then cells were collected for detecting cytokine expression by real-time PCR.The results showed that the expression level of IL-6,IL-8,TNF-?and RANTES were up-regulated in the cells treated with N-exosomes.