Exosome-Mediated Microrna Transfer Between Porcine Cells And Its Inhibitory Effect On Virus Replication

Exosomes are eukaryotic cell-derived microvesicles, which contain a variety of bioactive molecules and play important roles in material and information exchanges between cells. Recent studies have shown that exosomes also play an important role in small interfering RNA (siRNA) and microRNA (miRNA) transfer between human and murine cells, but the exosome-mediated siRNA or miRNA transfer between porcine cells has not been reported.To study the role of exosomes in artificial microRNA (amiRNA) transfer between porcine cells, in the present study two recombinant adenoviruses, namely rAd-amiR3UTR-EIRES-GFP and rAd-amiRcon-EIRES-GFP, which expressed the artificial miRNA (amiRNA) targeting3’-untraslated region (3’-UTR) of porcine reproductive and respiratory syndrome virus (PRRSV) or a control amiRNA, were generated. Typical morphology of adenoviruses was confirmed by electron microscopy. The presence of the amiRNA expression cassette in the viral genome was confirmed by PCR detection. Expression of the amiRNA in the virally transduced cells was detected by RT-PCR.Pig kidney cell line PK-15and porcine alveolar macrophage (PAM) cell line3D4/21, as well as primary PAM, were infected with rAd-amiR3UTR-EIRES-GFP or rAd-amiRcon-EIRES-GFP. The cellular RNA was extracted and the amiRNA expression was detected by RT-PCR. On day3post infection, the cells and supernatants were collected, and exoxomes were purified from the cell supernatants for semi-quantitative detection of amiRNA expression. PAM cells were incubated with PK-15-or3D4/21-derived exosomes and then infected PRRSV. At24h post infection, cells were analyzed by immunofluorescence and the virus was harvested for viral titration on MARC-145cells. The results showed that both recombinant virally infected PK-15and3D4/21cells could express the sequence-spcific amiRNAs, which could be sereted into the cell medium via exosomes. After co-incubation with PK-15or3D4/21cell-derived exosomes, PRRSV titer in PAM cells was reduced by1.54or1.13log TCID50. These data suggest that the amiRNA-containing exosomes could be secreted from and taken up by porcine cells, which could act as an important mechanism for delivery of anti-viral interfering RNAs.