Effect And Mechanism Of Golgi To ER Retrograde Trafficking On The Transport And Proliferation Of Classical Swine Fever Virus

Classical swine fever(CSF),a highly contact infectious disease,caused by Classical swine fever virus(CSFV)infecting domestic pigs or wild boars,which has caused huge economic losses to the pig industry all over the world.CSF is listed as one of the notifiable swine diseases by the Office International Des Epizooties(OIE)and designated as one of the class A animal disease in China.Although vaccine inoculation and harmless treatment of sick pigs have played a positive role in the prevention and control of CSF in China.However,CSF still occurs from time to time,especially recessive infection and persistent infection,which restricts the healthy development of pig industry in China.The effective infection of virus is the basis of the disease.The virus enters the proliferative sites and assembles replicationrelated components to effectively establish infection and then cause disease.Intracellular vesicle transport system plays an important role in virus life cycle,such invasion,assembly and virion release.Discovering and revealing the relationship between these vesicle transport systems and CSFV infection is the basis for elucidating the pathogenic mechanism of CSFV.Therefore,this research mainly focuses on the effect and mechanism of Golgi-ER retrograde trafficking mediated by COP?-coated vesicles system and Golgi-ER retrograde trafficking mediated Rab18 on the proliferation of CSFV in swine umbilical vein endothelial cell lines(SUVECs).The following results were obtained:1.COP?-coated vesicles were involved in the transport of CSFV virion,and in CSFV proliferation.(1)The laser confocal assay showed that COP?-coated vesicles obviously co-localized with CSFV E2 protein and C protein,which proved that COP?-coated vesicles were used for CSFV intracellular transport.(2)Knockout cell lines against COP?? and ? subunits were established.RT-q PCR and laser confocal assays showed that CSFV entry were significantly impaired in COP?-coated vesicles knockout cells(P<0.05 or P<0.01),proving that the COP?-coated vesicles were involved in CSFV transport in entry steps.(3)The laser confocal assays showed that CSFV obviously co-localized with Rab5 and Rab7 in the stage of virus entry,and CSFV production was significantly impaired in Rab5 and Rab7 interfering cells(P<0.05 or P<0.01),which proved that CSFV was dependent on early endosome and late endosome to entry SUVECs.In addition,laser confocal assay showed that the co-localization of CSFV with Rab7 was disrupted in Brefeldin A(BFA)and Golgicide A(GCA)-treated cells,revealing the mechanism of COP?-coated vesicles transport CSFV particles through the late endosomal pathway.(4)CSFV infection was significantly impaired in COP?-coated vesicles inhibition cells by using inhibitors of BFA and GCA,or in COP?? and ? subunits knockout cell lines(P<0.05 or P<0.01).While,CSFV infection significantly promote the COP?subunits m RNA expression(P<0.05 or P<0.01),and disrupt the COP?-coated vesicles distribution.2.COP?-coated vesicles regulator of Rab1 b was required for CSFV infection.(1)Rab1b knockdown cell lines was established by package and infection of recombinant lentiviruses in SUVECs.RT-q PCR and IFA were used to detect the proliferation of CSFV in Rab1 b knockdown cell lines.The results showed that CSFV production was significantly impaired in Rab1 b knockdown cell lines(P<0.01 or P<0.001).In addition,recombinant plasmid of Rab1 b inactivating mutant(Rab1b-N121I)was constructed and transfected into SUVECs.RT-q PCR and IFA assays results showed that CSFV replication was significantly inhibited in Rab1b-N121I-transfected cells(P<0.05 or P<0.01).(2)In addition,confocal microscopy and Gaussia luciferase flash were used to detect Golgi apparatus and cell secretory in CSFV-infected cells.The results showed that CSFV infection disrupt the Golgi apparatus and block cell secretory pathway.3.COP?-coated vesicles regulator proteins of Golgi-specific BFA resistance factor 1(GBF1)and ADP-ribosylation factor 1(ARF1)were required for CSFV proliferation.(1)GBF1 knockdown cell lines was established by package and infection of recombinant lentiviruses in SUVECs.RT-q PCR and IFA were used to detect the production of CSFV in GBF1 knockdown cell lines.The results showed that CSFV proliferation was significantly inhibited in GBF1 knockdown cell lines(P<0.01 or P<0.001).Then,we constructed a series of plasmids expressing GBF1 truncation mutants fused to a Flag-tag and investigated their behavior during CSFV infection.Our data showed that the Sec7 domain of GBF1 was important for CSFV proliferation.(2)Small interfering RNA(si RNA)sequences against Sec7 domain of GBF1 effector molecules ARF1,ARF2,ARF3,ARF4,ARF5 were separately transfected into SUVECs.The results showed that CSFV production was significantly impaired in si ARF1,si ARF2,si ARF3,si ARF4 and si ARF5 transfected cells(P<0.01 or P<0.001),and found that ARF1 plays a major function in CSFV proliferation by regulating the mitochondrial cholesterol transport with a Sec7-dependent manner in CSFV infection.(3)We also found that CSFV infection induced m RNA expression of ARF1,ARF2,ARF3,ARF4 and ARF5(P<0.05 or P<0.01),but did not affect their intracellular localization.4.Rab18 protein participates in CSFV RNA replication,assembly and proliferation through interaction with NS5 A.(1)Rab18 knockdown cell lines were established by package and infection of recombinant lentiviruses in SUVECs.RT-q PCR and IFA assays found that Rab18 was required for CSFV RNA replication and assembly.(2)Co-immunoprecipitation(Co-IP),glutathione S-transferase pull-down(GST pulldown)and laser confocal assays showed that Rab18 mediated NS5 A intracellular trafficking by interacting,revealing the mechanism of Rab18 promotes CSFV RNA replication and assembly.(3)By regulating the expression of Rab18,it was found that the overexpression of Rab18 or the activated mutant(Rab18-Q67L)significantly promoted the CSFV proliferation(P<0.05 or P<0.01),and the knockdown of Rab18 or the overexpression of the inactivated mutant(Rab18-S22N)significantly inhibited the CSFV proliferation(P<0.05 or P<0.01),indicating that Rab18 positively regulates the proliferation of CSFV.In conclusion,we found that COP?-coated vesicles and its mediators of Rab1 b,GBF1and ARFs were involved in CSFV infection.COP?-coated vesicles transport virion through the late endosome during the CSFV entry steps,and the key regulatory protein ARF1(Sec7domain dependent)promotes the CSFV production by regulating mitochondrial cholesterol transport.It was proved that the COP?-coated vesicle-dependent Golgi-ER retrograde trafficking was an important host factor for the proliferation of CSFV.In addition,Rab18 regulates the CSFV NS5 A intracellular transport through interaction,and participates in CSFV RNA replication,progeny virus assembly and proliferation.It was proved that Rab18 mediated Golgi-ER retrograde trafficking was required for CSFV proliferation.These results provide a novel theoretical basis to elucidate the role and mechanism of Golgi-ER retrograde trafficking in CSFV infection.