Visualization Of Ebola Virus Invasion Of Host Cells

Ebola virus is a potent hemorrhagic fever virus,which causes Ebola hemorrhagic fever in human,the mortality rate is as high as 90%.The pathogen causing this infectious disease is the Ebola virus,a non-segmented single-stranded negative RNA virus,which belongs to the Filamentous virus family,the Ebola virus genus.Under the electron microscope,the Ebola virus shows a filamentous structure with diameter of about 80nm and the length of 300?1500nm.Ebola virus structural proteins include nucleoprotein NP,envelope protein GP,VP30,VP35,matrix proteins VP24 and VP40,and L(RNA-dependent RNA polymerase).NP is the main component of the nucleocapsid,with VP30,VP35 and L together constitute a nuclear protein complex(RNP complex),responsible for the replication and transcription of the virus.Virus-like particles are empty or encapsulated particle structures that do not contain viral genomes.In this study,we constructed and expressed the Ebola virus-like particles(VLP)and the replication and transcription-competent Ebola virus-like particles(trVLP),and further fluorescently labeled VLP and trVLP with the recombinant fluorescent protein technology and the specific protein fluorescence dye,respectively.The fluorescently labeled viral particles provided a basis for the real-time dynamic study of the mechanism for Ebola virus invasion.This study comprises two parts of work:1.Expression and identification of Ebola virus VLP/trVLPThe Ebola virus-like particles(VLP)assembled with Ebola virus matrix protein VP40 and GP.The replication and transcription-competent Ebola virus-like particles(trVLP)were constructed with VP40.VP24 and glycoprotein GP as the main body of the package,and with other proteins VP35,VP30,NP,L to express virus-like particles with transcription and replication capabilities.We observed the filamentous structure of VLP/trVLP similar to natural Ebola virus by transmission electron microscopy,which also morphologically demonstrated the successful expression of virus-like particle.For trVLP containing a reporter gene,we used the luciferase detection system to detect the expression of the reporter gene,and the positive detection value confirmed the successful expression of trVLP.In addition,the Ebola VLPs with recombinant expressed green fluorescent protein and the trVLP labeled with the fluorescent protein dye could be observed under the fluorescence microscopy.Under the fluorescence microscope,it was observed that the virus-like particles could adhere and enter into the cells,which proved that the VLP and trVLP we constructed had the ability to enter the cells.2.Study the interaction between Ebola virus and cell membrane lipid raftUnder the PE UltraVIEW VoX two-disc live cell fluorescence confocal microscopy,set with multiple channels,we can stimulate the fluorescent signal in the same field of view of different channels.Through the use of these technologies,we could study the Ebola virus in the process of invading cells.We found that Ebola virus-like particles(VLP)and replication and transcription-competent Ebola virus-like particles(trVLP)could adhere and enter the cells,and both of them had different degrees of co-localization with the lipid raft components on the cell membrane.Real-time dynamic studies found that cell membrane lipid raft played an important role in the process of Ebola virus entering cells.The cells were treated with the lipid raft inhibitor(methyl-?-cyclodextrin)before the adsorption of viral particles.The efficiency of entering the cells showed a significant decline under the treatment of inhibitor,indicated that the lipid raft played an important role in the entering process.Therefore,we found that the Ebola virus entered cell through the lipid raft path,providing a basis for identification of new drug targets.