The Role And Mechanism Of Lipid Rafts In Enterovirus D68 Infection

Enterovirus D68(EV-D68)belongs to the enterovirus genus of the picornaviridae family and belongs to a single-stranded positive-stranded RNA virus without an envelope.It was first isolated from children's oropharyngeal swabs in California in 1962.It can cause neurological diseases and respiratory diseases in children,and respiratory diseases are more common in the clinic.In recent years,EV-D68 has erupted in many countries or regions around the world,and currently there are no specific drugs and vaccines.Lipid raft is a microdomain rich in sphingolipid and cholesterol on the cell membrane,and is widely distributed in the cell membrane system.Under the influence of internal and external factors,lipid rafts can dynamically and selectively recruit host proteins to participate in various biological functions of cells.When an enveloped virus infects a host,the invasion process is mainly the fusion of the virus envelope and the host cell membrane,and then the virus particles invade the host cell.Viral glycoprotein molecules mediate this fusion process by recognizing and binding the corresponding specific receptors.Lipid rafts are found in many viruses such as HIV-1,influenza virus and human herpes virus-6),Etc.play an important role in the intrusion,assembly and release process.When the virus binds to the host cell membrane receptor,it also activates related cell signaling molecules coupled to the receptor,thereby activating related signal pathways to participate in various physiological processes of the host cell,and some viruses participate in the correlation through lipid raft Signalling.The role of lipid rafts in EV-D68 infection has not been reported in the relevant literature.In order to investigate the role of lipid rafts in EV-D68-infected cells,and finally to provide a basis for elucidating the molecular mechanism of EV-D68's pathogenesis and finding new antiviral targets,we completed the following studies.1.Using methyl-?-cyclodextrin(M?CD)to remove part of the cholesterol molecules that maintain the stability of the lipid raft to destroy the lipid raft structure of the host cell membrane can inhibit the penetration of EV-D68 and reduce the virus titer.The supplementation of exogenous cholesterol during virus penetration can reverse the inhibition of EV-D68 penetration and replication by the removal of cell membrane cholesterol by M?CD,indicating that lipid rafts are closely related to EV-D68 penetration and replication.2.The effect of lipid rafts on virus adhesion and entry was detected using qRT pcr.It was found that the destruction of lipid rafts inhibited the entry of viruses,and the effect of recovering the entry of viruses was restored after filling with exogenous cholesterol.3.Western blot was used to detect the extracted lipid raft components.It was found that EV-D68 VP1 and lipid raft marker proteins Caveolin-1 have co-localization,which proves that EV-D68 can enter cells through the lipid raft structure.4.M?CD can disperse cellular lipid raft components,and its marker proteins,viruses,and non-lipid raft region marker proteins have co-localization.EV-D68 will entry cells through non-lipid raft regions.5.Confocal microscopy detected that after M?CD treatment of cells,vesicle acidification was affected during virus entry.6.Destroying lipid rafts inhibits the infection of the EV-D68 epidemic strain.In summary,this study found that lipid rafts are involved in the penetration of EV-D68 and acidification of endosome vesicles,and the formation of endosome vesicles may be affected after the lipid rafts are disrupted,interfering with the release of the viral genome and thus the virus life cycle.After the lipid raft is destroyed,EV-D68 enters the cell through the non-lipid raft region.