Exosomes Carrying Micro RNAs Promote Newcastle Disease Virus Replication

Exosome(Exo)is a kind of small vesicles released to the extracellular space secreted actively by cells.The diameter of the vesicles is about 30-150 nm.It plays a key role in intercellular communication by transmitting proteins,Ribonucleic acid(RNA),lipids and other bioactive substances between cells.In recent years,quite a lot of research reports about exosomes and viral infection have been carried out,especially the biological function of exosomes in the study of Human Immunodeficiency Virus(HIV)and the Hepatitis C virus(HCV)infection.However,there are still no reports about exosomes in Newcastle disease virus(NDV)infection.Micro RNA(mi RNA),as a member of the non-coding RNA(nc RNA)family,regulates negatively targeted genes through cleaving m RNA and inhibiting protein translation and participates in many physiological and pathological processes.In virus infection,a variety of micro RNA has been found to be able to regulate virus replication,but the mi RNA related to NDV infection is still rarely reported.Obtaining high-quality exosomes samples is the first step in the study of exosomes and NDV infection.To acquire the sample,we isolated and identified the reliable and effective exosomes secreted by He La cells by the differential centrifugation method,transmission electron microscope(TEM),Nanosight Tracking Analysis(NTA),Western blotting(WB),respectively.And we found obvious cell lesion in DF-1 cells incubated with purified exosomes secreted by NDV-infected He La cells compared to the control groups,suggesting that the exosomes can enhance NDV infection on He La cells.Microarray was used to detect mi RNAs expression difference in exosomes derived from NDV-infected and uninfected He La cells.It was found that the expression difference of 236 host mi RNAs had changed more than two times,of which 145 mi RNAs showed a significant upregulation.25 mi RNAs possible targeting interferon-?(IFN-?)were predicted with the bioinformatics software.He La cells were treated with poly(I:C)or infected with NDV and 25 mi RNAs overexpressed in these cells,respectively.IFN-? m RNA and protein levels detected with RT-q PCR are significantly decreased when three of 25 mi RNAs(mi R-1273 f,mi R-1184,mi R-198)overexpressed.The results detected by luciferase reporter system was shown that the fluorescence intensity decreased significantly when mi RNA mimics and luciferase vector containing IFN-? 3' UTR were transfected into H293 T cells,suggesting that these three mi RNAs may target IFN-? 3' UTR directly.Later three mi RNAs were found that they can not only affect interferon-stimulated genes(ISGs)transcription such as Interferon-stimulated gene 15(ISG15),Interferon-stimulated gene 56(ISG56),antiviral Myxovirus resistance protein 1(Mx1),the oligoadenylate synthetase 1(OAS1),but also enhance NDV NP m RNA transcription and NDV NP protein translation.In addition,it was found that exosomes carrying mi RNAs could enhance the plaque formation on DF-1 cells during NDV infection in the process that mimics exosomes carrying mi RNAs,suggesting that these exosomes could promote NDV spread.In short,we infected He La cells with NDV infection.It was found that three mi RNAs(mi R-1273 f,mi R-1184 and mi R-198)could affect NDV replication through directly targeting IFN-? in a series of experiments.More importantly,this was the first time it has been demonstrated that exosomes could promote NDV infection and carry host mi RNAs to affect neighboring cells resulting in improving the susceptibility of these cells to NDV and promoting virus infection,suggesting that exosomes carrying mi RNAs for virus replication may be a new way to promote NDV infection.