Study Of The Transport And Transmission Of Classical Swine Fever Virus Through Coatomer ProteinⅡ-Autophagosome-Extracellular Vesicles

Classical swine fever（CSF）is a highly contagious disease of pigs and wild boars caused by classical swine fever virus（CSFV）,which seriously restricts the development of pig industry.In China,a combination of compulsory vaccine immunization and elimination of diseased pigs has been used to stamp out CSF,but in recent years,recessive infections and chronic persistent infections have brought new challenges in CSF prevention and control.A comprehensive understanding of the proliferation and pathogenic mechanisms of CSFV will help to stamp out CSF.For many years,scientists have focused on the invasion,genome replication and immune evasion of CSFV,but little is known about the processes of the CSFV progeny virions assembly,intracellular transport,and intercellular transmission.Therefore,in this study,we analyzed the roles of coatomer proteinⅡ（COPⅡ）and cellular autophagy pathway in the intracellular transport and release of CSFV,explored the diversity of CSFV transmission modes in cultured cells,and identified the transmission mediator,which revealed the mechanisms of which CSFV utilizes COPⅡ-autophagosome-extracellular vesicles（EVs）to transport and transmission.The following results were obtained.（1）CSFV particles traffic through COPⅡ-mediated transport pathway to export from endoplasmic reticulum（ER）.The differential expression of COPⅡproteins in spleen,kidney,tonsil,and intestine of CSFV-infected piglets was detected by immunohistochemistry（IHC）and Western blot,and the results showed that CSFV infection promoted the expression of Sar1b,Sec23,Sec13 and Sec31A.Inhibition of COPⅡassembly and transport by Sar1b mutation revealed no significant changes in viral RNA replication in the first life cycle after CSFV infection,but the infectivity and titer of progeny viruses decreased significantly（P<0.01）.The laser confocal assay showed that the mutation of Sar1b increased the co-localization of CSFV E2 protein with the ER;Transmission electron microscope（TEM）showed that CSFV particles gathered on the outer surface of the ER in control cells.The activate mutation of Sar1b increased the number of virus particles gathered on the ER,and the inactive mutation blocked the assembly of virus particles in the ER,suggesting that COPⅡis involved in the assembly and transport of CSFV.Finally,viral particles were detected in the COPⅡvesicles isolated by immunoprecipitation,which indicated that COPⅡvesicles are the carriers for CSFV particles export from the ER.（2）CSFV enters COPⅡvesicles via capsid protein binding Sec24A.A biotin proximity labeling（P L）method was developed to capture COPⅡcomponents and cargoes by fusion expression of Sar1b with biotin ligase.The specificity of Sar1b-Bio ID2-mediat ed PL for l abeling COPⅡcomponents and cargos was verified by a streptavidin pull-down assay and a strept avidin fluorescence assay.Using PL,we analyzed the dependence of CSFV protei ns on COPⅡm ediated transportation,and revealed that viral structural protein C,E^{r n s},E2,and nonstructural protein NS5A were associated with COPⅡtransport.Using laser confocal assay to detect the localization of C,E^{r n s},E2,NS5A with COPⅡproteins,we found that the envelope prot ein E2 an d capsid prot ein C were obviously co-l ocali zed with Sec24A.Moreover,the result s of co-immunoprecipitation（Co-IP）showed that capsid protein C bind to Sec24A,indicating that CSFV capsids enter COPⅡvesicl es by binding Sec24A.（3）The cellular autophagy pathway is involved in the intracellular transport and release of CSFV.Using sh RNA and Sar1b mutation to inhibit COPⅡtransport,we found that the level of CSFV-induced autophagy was decreased significantl y（P<0.05）.The autophagy activator Rapamycin promoted the release of CSFV,whereas the inhibitors 3-methyladenine（3-MA）and Wortmannin inhibited CSFV release.Meanwhil e,suppression of the autophagy proteins BECN1 and LC3B expression by sm all int erfering RNA（si RNA）resulte d in a significant reduction in CSFV rel ease level（P<0.01）,indicating that the release of CSFV is regulated by the autophagy pat hway.TEM observation showed that CSFV particles aggregated in autophagy like vesicles,indicating that autophagosomes were in volved in CSFV intracellular transport.Chloroquine（CQ）,an acidic lysosomal neutralizer,significantly inhibited the CSFV release.Using laser confocal assay,we detected the fluorescence distribution of the reporter plasmid m Cherry-GFP-LC3B,the co-localization of autophagosome and lysosome,and the co-localization of CSFV and lysosome in CSFV infected cells,which revealed that CSFV distributed in lysosomes and induced the fusion of autophagosom es and lysosomes,indicating that the formation of autolysosomes invol ves in the transport and release of CSFV.（4）CSFV hijacks the EVs originated from autophagy to transfer virions between cells in a neutralizing antibody-resistant manner.Utilizing low-melting point agarose and neutralizing antibodies to limit the viral free spread,and comparing the efficiency of viral transmission and the rate of cell proliferation,we found that CSFV transmitted from infected-to uninfected-cells in the cell-to-cell ways.Using Transwell to block the cell contact,repeated freeze-thawing of viral supernatant to destroy the vesicular structure,differential centrifugation to isolate EVs,and TEM observation,CSFV was found to release and spread viral particles through EVs.The CSFV-containing EVs were purified by using gel filtration chromatography（GFC）,and found to contain autophagy proteins LC3B and BECN1.At the same time,the LC3B-positive EVs were isolated by immunoprecipitation,and identified to contain CSFV RNA and envelope protein,and have CSFV infectivity by multiple assays,indicating that CSFV-containing EVs are originated from autophagy.Through activating or inhibiting the autophagy pathway by autophagy regulators and si RNA,the infectivity of EVs released from CSFV infected cells was found to be enhanced with the activation of the autophagy pathway and decreased with the inhibition of the autophagy pathway,indicating that EVs mediated CSFV release and transmission are regulated by cellular autophagy.In summary,this study found that CSFV utilizes the COPⅡmachinery to form infectious particles in the ER.The viral particles enter COPⅡvesicles to exit the ER through early secretory pathway,and then traffic to autophagosomes and autolysosomes,and finally exit host cells via EVs and spread to other cells.These findings contribute to the understanding of CSFV transport,release,and transmission,and provide new scientific data for clarifying the proliferation and pathogenic mechanism of CSFV.