Molecular Mechanism Of ESCRT Regulating CSFV Infected PK-15 Cells

Classical swine fever(CSF)is a viral infectious disease caused by classical swine fever virus(CSFV),which has a high morbidity and mortality,and has brought huge economic losses to the world pig industry.Endosomal sorting complex required for transport(ESCRT)is a significant molecular machine for the sorting,transport and degradation of membrane proteins in eukaryotic cells,participating in multiple stages of the life cycles of enveloped viruses.Firstly,this study intends to use si RNA,RT-q PCR,confocal microscopy,Co-IP and TEM assays to investigate the molecular mechanisms of ESCRT subunits regulating CSFV invasion into porcine kidney cells(PK-15)by interacting with clathrin and a variety of endosomal proteins.In addition,we further elucidated the molecular mechanisms of ESCRT subunits interacting with the CSFV nonstructural(NS)proteins to induce the formation of virus replication complex(VRC)on endoplasmic reticulum(ER)membrane and then promoting virus replication.Finally,our study also found that CSFV budding depended on the participation of ESCRT subunits.In conclusion,this study clarified the specific molecular mechanisms of ESCRT subunits involving in regulating multiple life cycles of CSFV infected PK-15 cells,which laid a theoretical foundation for the prevention and control of CSFV infection.This research will be carried out from the following three aspects:1.Molecular mechanisms of ESCRT regulating CSFV invasion of PK-15 cellsIn general,the life cycles of virus can be divided into: adsorption,entry,uncoating,biosynthesis,assembly and release.Invasion is an indispensable part for virus crossing the cell membrane barriers and entering the host cell,and it is also a key step to determine whether it can successfully infect cells.Furthermore,in order to analyze whether CSFV invasion into PK-15 cells depends on the participation of ESCRT subunits,this study clarified the molecular mechanisms of ESCRT subunits mediated virus invasion based on si RNA,RT-q PCR,confocal microscopy,Co-IP,dominant negative mutant and other technologies.In this study,through si RNA experiment,we found that Tsg101,EAP20(VPS25),CHMP4 B and CHMP7 subunits plays an important role in the entry of CSFV,and VPS37 A,VPS37B,CHMP1 A,CHMP1B,CHMP2 B subunits also plays a significant role in the invasion of CSFV.In addition,after CSFV infection,only the expression level of endogenous Tsg101 subunit was significantly enhanced,while the other endogenous ESCRT subunits had no significant changes,and overexpression of exogenous Tsg101 protein would also promote the invasion of CSFV.Finally,our results revealed that Tsg101,VPS25,CHMP4 B and CHMP7 subunits colocalized with virus particles in the early stage of CSFV invasion through Co-IP and confocal microscopy experiment.However,VPS4 A subunit did not colocalized with CSFV.More importantly,Tsg101,VPS25,CHMP4 B and CHMP7 subunits first mediated the endocytosis of CSFV by interacting with Clathrin,and then sequentially interacted with early endosomes(Rab5),late endosomes(Rab9)and lysosomes(LAMP-1)to complete the transport process of virus particles.In conclusion,this study revealed the molecular mechanisms of ESCRT subunits regulating CSFV invasion into PK-15 cells by interacting with endosomal proteins such as Clathrin,Rabs and LAMP-1 for the first time,which providing rich theoretical basis for the study of ESCRT subunits involved in the regulation of flavivirus invasion mechanism.2.Study on the interaction between ESCRT subunits and CSFV proteinsVirus replication is an indispensable key link in viral life cycle.In this process,the virus interacts with host factors to induce the change of cell structures,which providing a platform for the replication of virus particles.In order to analyze whether the ESCRT subunits participating in the replication and budding of CSFV,and the interaction mechanisms between the ESCRT subunits and viral proteins during the replication process.Firstly,we infected CSFV with different MOIs after interfering with the protein expression of endogenous ESCRT subunits in PK-15 cells,and the results revealed that HRS,Tsg101,VPS28,EAP20,CHMP2 B,CHMP4B,CHMP7,VPS4 A and ALIX subunits played a crucial role in the replication process of CSFV.VPS37 A,VPS37B,EAP45,CHMP1 A,CHMP1B,CHMP4 A and CHMP4 C subunits also showed a significant effect in the replication of CSFV.Furthermore,our studies also illustrated that HRS,Tsg101,VPS28,EAP20,CHMP1 B,CHMP2B,CHMP4 B,CHMP7,VPS4 A and ALIX subunits also played significant roles in the budding process of CSFV.VPS37 A,VPS37B,EAP45,CHMP1 A,CHMP4A and CHMP4 C subunits also affecting virus budding.In addition,after infected with CSFV,only the expression level of endogenous Tsg101 subunit in cells was significantly enhanced,while the other endogenous ESCRT subunits had no significant changes,and overexpression of exogenous CSFV protein had no significant effect on the expression level of other endogenous ESCRT subunits.Finally,this study also revealed that HRS,Tsg101,VPS28,EAP20,CHMP2 B,CHMP4B,CHMP7,VPS4 A and ALIX subunits colocalized and interacted with the the NS proteins of CSFV by confocal microscopy and Co-IP experiment,and CSFV infection can also promote the mutual recruitment of ESCRT subunits.In summary,this study systematically analyzed the molecular mechanisms that virus infection can promote the mutual recruitment of ESCRT subunits,and these ESCRT subunits can promote virus replication by interacting with CSFV NS proteins,which provides a richer theoretical basis for the study of CSFV life cycle.3.ESCRT subunits promotes virus replication by affecting the formation of VRCDuring virus infection,the virus usually utilizes the components of the host cell membrane and host factors,induces changes in the organelle structure to form a special replication compartment in the endoplasmic reticulum region,namely the virus replication complex(VRC).Firstly,based on the TEM technology,our study found that PK-15 cells infected with CSFV could induce the formation of VRC in the ER region.Moreover,our study revealed that HRS,Tsg101,VPS28,VPS25,CHMP2 B,CHMP4B,CHMP7,VPS4 A and ALIX subunits were significantly colocalized with ds RNA by confocal microscopy experiment,which indicated that these ESCRT subunits were involved in the formation of VRC during CSFV replication.In addition,we infected PK-15 cells with different MOIs of CSFV,and then extracted the total protein of the VRC,it was also confirmed that these ESCRT subunits were significantly enriched in VRC after virus infection,and showed an obvious positive correlation trend with infection dose.Finally,our study also found that these ESCRT subunits were also colocalized with CSFV NS proteins and ER,but not Golgi,indicating that the VRC involved in the formation of ESCRT subunits which locating in the ER region.Interestingly,our study found that VPS4 A subunit colocalized with lipid droplets(LDs)and VRC for the first time,suggesting that VPS4 A subunit may act as a bridge for the interaction between LDs and VRC during the formation of VRC.In conclusion,this study elucidates in detail the molecular mechanisms that ESCRT subunits induces the formation of VRC on ER by interacting with the virus NS proteins,and LDs act as the energy supply platform to promote virus replication.