Mechanism Of Action Of Exosomes In The Regulation Of Tembusu Virus Infection

Tembusu virus(TMUV)infection is an emerging infectious disease that has been affecting waterfowl farming in China since 2010.TMUV infection is characterized by a significant drop in laying duck and neurological symptoms in ducklings.As a member of the genus Flavivirus,TMUV has a wide range of hosts,including mosquitoes,wild birds and even humans,posing a huge challenge to public health security in China.Genetically engineered,weakened and inactivated vaccines have been developed,while there are still many problems in the existing vaccines such as ineffective immunization and poor immune protection.Studies have shown that exosomes are one of the tools used especially by RNA viral infection.Both exosomes and cell membranes have a lipid bilayer that protect their contents from degradation,which can maintain their biological activity even after being transported over long distance and periods of time.Additionally,bilayer can also help exosomes escape from immune surveillance,which is more conducive to the viral diffusion.Therefore,understanding the relationship between TMUV and exosomes,as well as the role of exosomes in the process of TMUV infection can lay the theoretical foundation for the prevention and control of TMUV infection,and the development of novel biologics.The main research contents are as follows.1.Isolation and identification of exosomes released from TMUV-infected cellsDue to the overlapping particle size distribution of TMUV and exosomes,exosomes and viruses cannot be separated with commonly used separation methods.Therefore,we used ultracentrifugation combined with to CD63 immunomagnetic beads purification method to acquire purified TMUV-exosomes from HD11 and HEK293 cell lines.A series of characterizations of exosomes were identified by nano-flow technique to detect the particle size distribution range of exosomes,Western Blot to detect surface markers of exosomes and TEM to observe the morphology of exosomes.The results showed that exosomes with high purity could be isolated with this method,which were cup-shaped,with particle size distribution of30-200 nm,positive for exosome surface markers and negative for endoplasmic reticulum markers,and could be separated from free viral particles without TMUV contamination.2.Effect of TMUV infection on exosome releaseTo further understand the effect of TMUV infection on exosome release,we detected the exosomal marker TSG101 by Western Blot combined with nano-flow detection analysis and confirmed that TMUV infection significantly promoted exosome secretion from HD11 and HEK293 cells.In addition,TMUV-exosomes contain the whole genomic sequences of TMUV,as well as pr M?E?NS1?NS2B?NS4B and NS5 protein by viral nucleic acid detection and protein profiling.Furthermore,we used three monoclonal antibodies of TMUV to confirm that E?pr M and NS5 protein exist in TMUV-exosomes indeed.In addition to the viral components,we identified a total of 5360 host proteins in TMUV-exosomes and Mock-exosomes using labelfree quantitative proteomics techniques,with 52 proteins up-regulated and 130 proteins downregulated.These host proteins are involved in endocytosis,vesicular transport,viral infection and cellular autophagy,indicating that TMUV infection causes up-and down-regulation of host proteins,which are thus enveloped into exosomes with widely different compositions.3.Effect of exosomes on TMUV replicationTo further analyze the role of TMUV-exosomes in TMUV infection,PKH67-labelled mock-exosomes and TMUV-exosomes were co-incubated with HEK293 and HD11 cells,which demonstrated that TMUV-exosomes could be actively taken up into cells by laser confocal.After inoculation of equal amounts of TMUV and TMUV-exosomes into cells separately,it was found that TMUV-exosomes could establish proliferative infection in recipient cells,while TMUV-exosomes were less efficient in infecting HEK293 cell lines.After co-incubation of exosomes extracted at different time points with cells,it was found that exosomes extracted at12 h and 24 h significantly promoted TMUV replication,and TMUV-exosomes suppressed the expression of ISGs and TNF.Exosome secretion inhibition assay revealed that GW4869 significantly inhibited the production of exosomes in HEK293 cells and significantly inhibited the production of TMUV.By extracting exosomes from TMUV-infected cells treated with 10?M GW4869,it was shown that the TMUV proteins contained in the TMUV-exosomes was significantly reduced,as well as the infectivity to cells,indicating that inhibition of exosome secretion significantly inhibited the replication and spread of TMUV.In addition,to investigate whether TMUV-exosomes can be transmitted without receptors,we performed the receptorclosure assay,which showed that the infectivity of TMUV was significantly reduced with receptor closure,while the infectivity of TMUV-exosomes to cells was not affected,thus confirming that TMUV-exosomes can mediate infection via a receptor-independent pathway.4.Study on the mechanism of TMUV-exosomes promoting TMUV replicationTo investigate the relationship between TMUV-exosomes and TMUV replication,we screened the Rab27 a protein,which was associated with exosome secretion and transport in the preliminary label-free quantitative proteomics results,to determine its role in exosome and TMUV replication.We found that TMUV infection significantly promoted the expression of Rab27 a.And then,overexpression of Rab27 a protein could also significantly promoted TMUV replication.Additionally,the TMUV proteins in exosomes was also significantly increased by overexpressing Rab27 a protein.In order to determine which protein component of TMUV could promote exosome release,the expression of Rab27 a and TSG101 was detected by q RTPCR after overexpression of ten proteins of TMUV,and the expression of TSG101 was detected in NS4A-overpression exosomes.The results showed that TMUV NS4 A protein could significantly promote the release of exosomes.These results showed that TMUV infection promoted the release of exosomes,while TMUV-exosomes could in turn promote TMUV replication.TMUV replication was also significantly inhibited with GW4869,and TMUV-exosomes could mediate TMUV receptorindependent infection.In the interaction between TMUV and TMUV-exosomes,the TMUVNS4 A protein and the host Rab27 a protein play an important role,that is the NS4 A protein promotes the release of exosomes encapsulated with TMUV nucleic acid and proteins by upregulating Rab27 a expression,which in turn delivers viral and host components to surrounding or distant uninfected cells,which promotes the transmission and spread of TMUV.