Effects And Mechanisms Of Rab1A And Rab5 On The Proliferation Of Classical Swine Fever Virus

Classical swine fever(CSF),caused by classical swine fever virus(CSFV),is a high pathogenicinfectious diseasecharacterized byhigh fever and organization hemorrhage.CSF,one of the diseases notifiable to the OIE(www.oie.int),is classified as the 1st animal disease in China and causes potential financial losses in the pig industry globally.CSFV is asingle-stranded,positive-sense,enveloped RNA virus and belongs to the genus Pestivirus within the family Flavivridae.The viruses are strict intracellular parasite,due to their limited genome,viruses usually employ host factorsto complete their life cycles such as cell entry,genesynthesis,virions assembly and release.Thus,it is important for understanding the pathogenesis of the virus to explore the mechanism for cytokines regulating viral proliferation.There are large numbers of membrane organelles composed oflipid and protein in eukaryotic cells,and the vesicular transport system is responsible for the transport of intracellular substances between the membrane organelles.Rab protein belongs to the Ras small GTPase superfamily,is the largest subfamily involved in vesicular transport in the Ras superfamily and works in regulating the transport between the membrane organelles.Enveloped viruses guide their structural proteins and genomes to viral replication,assemblyand budding sites in a vesicle transport dependent manner.Rab protein has been shown to be involved in the proliferation of numerous important pathogenic viruses.In this study,we investigated the effects and mechanismsof Rab1 A and Rab5 on the proliferation of CSFV and the results are as follow:(1)The influence of Rab1 A on CSFV proliferation.Three short hairpin RNA were separately inserted into interference lentivirus vector,and the packaged recombinant lentiviruses were used to infect PK-15 cells to get three cell lines(PK-Rab1A-sh1,2,3)with Rab1 A knockdown.Analyses of the mRNA and protein levels of the three cell lines showed that PK-Rab1A-sh2 cells have the highest efficiency at inactivating Rab1 A.Analysis of CSFV proliferation in PK-Rab1A-sh2 cells showed that virus gene copies and E2 protein levels were significant lower than those from control cells,which indicated that Rab1 A depletion inhibits proliferation of CSFV.Besides,block of the Rab1A-regulated ER to Golgi transport was also showed to decrease CSFV proliferation.These results suggested that Rab1 A and Rab1A-regulated ER to Golgi transport are required for CSFV proliferation.(2)Rab1A promotes the assembly of CSFVvirions.RT-qPCR,IFA and Western blot were separately used to analysis virus gene copies,virus titers and virus E2 protein levels within the first lifecycle of CSFV.Results revealed that Rab1 A depletion decreased intracellular and extracellular CSFV titers,but did not affect intracellular virus genome copies and E2 protein expression,which suggested that Rab1 A is required for CSFV particle assembly rather than for genome replication or virion release.This conclusion was proofed by blocking the spread of virus using neutralizing antibodies,through which the negative effects of Rab1 A knockdown on multi-cycle replication of CSFV were eliminated.Moreover,co-immunoprecipitation and confocal microscopy assays showed that Rab1 A bound to CSFV NS5 A protein,indicating that Rab1 A and viral NS5 A proteins may work cooperatively during CSFV particle assembly.(3)The influence of Rab5 on CSFV proliferation.PK-15 cell lines with Rab5 overexpressing(PK-LV-Rab5)or knockdown(PK-Rab5-sh1,2,3)were constructed using recombinant lentivirus infection approaches.RT-qPCR and Western blot analyses showed that PK-LV-Rab5 cells overexpress Rab5 rightly and PK-Rab5-sh1 cells have the highest efficiency at inactivating Rab5.Analysis of CSFV proliferation in these two cell lines revealed that Rab5 overexpression significantly promotes CSFV proliferation,while knockdown of Rab5 significantly inhibits CSFV proliferation.Besides,eukaryotic plasmid mediated transient overexpression of Rab5 was also showed to enhance proliferation of CSFV.These results indicated that Rab5 positively regulates proliferation of CSFV.(4)Rab5 interacts with NS4 B to facilitate the formation of NS4B-related complex.The interaction between Rab5 and NS4B(one of CSFV replicase protein)was confirmed by co-immunoprecipitation and glutathioneS-transferase pulldown assays.Laser confocal microscopy assays further observed the co-localization of Rab5 and NS4 B.Furthermore,intracellular distributionof NS4B-Red presented as dot-like pattern in CSFV infectedPK-15 cells.The punctatesignal of NS4B-Red was disrupted when Rab5 function was inhibited by Rab5S34N(dominant negative mutantof Rab5),which indicated that Rab5 is essential to the formation of the NS4B-related complex.Moreover,viralNS3 and NS5 Awere observed to co-localize withNS4 Bin thecytoplasm,indicating that NS3 and NS5 A are components of the NS4 B relatedcomplex which should be the replication complex of CSFV.Collectively,this study illustrated that Rab1 A and Rab5 positively regulate proliferation of CSFV.Rab1 A binds to viral NS5 A protein and promotes assembly of virions to produce infectious viral particles.Rab5 interacts with viral NS4 B protein to facilitate the formation of NS4B-induced complex,which promotes proliferation of CSFV.