Preliminary Study Of Endocytosis Pathway Used By Rabies Virus

Rabies virus(RABV)is a highly lethal neurotropic zoonoses with a mortality rate of 100 percent.Once infected,RABV cause encephalomyelitis.The clinical manifestation includes fearing of water and wind,muscle spasm ect.RABV viral partical is composed of five structural proteins including nucleoprotein(N),phosphoprotein(P),glycoprotein(G),matrix protein(M)and large protein(L).N,P,L proteins consist ribonucleoprotein complex which plays an important role in viral replication and transcription.G is responsible for identifying the receptors on the cell membrane and mediating the intracellular trafficking process,and plays an important role in the process of virus entry into the cell.Endocytosis is the process of transporting extracellular substances into cells through the deformation of the plasma membrane.Not only macromolecules such as proteins and polysaccharides enter cells through endocytosis,but many viruses invade cells through endocytosis to achieve infection and multiplication.Many studies have shown that the enveloped virus enter host cells via receptor-mediated endocytosis,and it depends on many different endocytic proteins.Among them,clathrin is the most common marker protein,which mediates the endocytosis of most macromolecular substances.Caveolea also participates in various endocytosis processes and intracellular procedures.Caveolin-1 is a marker protein of the caveolea,and the stability of the caveolea requires the participation of cholesterol.Therefore,the cholesterol ratio in the cell membrane is a sign of measuring the function of the caveolea.To confirm the process of endocytosis,the proteinase k was added to N2 a cells at different virus incubation time point and then collected cell RNA.Then we take clathrin-and caveolae-dependent endocytosis as the starting point.Clathrin-specific inhibitor chlorpromazine,cholesterol extracted drug nystatin and methyl-?-cyclodextrin(M?CD),dynamin-specific inhibitor dynasore and vesicular acidification inhibitor bafilomycin a1 were added in N2 a cells,and then infected RABV.RT-q PCR and western blot were used to detect the expression level of RABV N.The rate of infection of RABV in N2 a cells was observed by immunofluorescence after drug treatment.Then si RNA was used to down-regulate clathrin heavy chain and caveolin-1 in N2 a cells.In order to verify the RABV endocytosis pathways in different cell lines,the mentioned inhibitors were added to SH-SY5 Y and Vero cells and then infected RABV.RT-q PCR was used to detect the RNA copy number of RABV N.Therefore,in this study,RABV spend 2h to enter N2 a cell.The results showed that RABV infection was significantly reduced after treatment with chlorpromazine,dynasore and bafilomycin a1 in N2 a cells.However,after treatment with nystatin and M?CD,there was no significant decrease in both expression of RABV N gene copy number and N protein.After knocking down of clathrin heavy chain expression in N2 a cell,the expression of RABV N was significantly reduced.But after knocking down the expression of caveolin-1,there was no significant decrease in the expression of RABV N.In addition,adding chlorpromazine,dynasore and bafilomycin A1 to SH-SY5 Y and Vero cells significantly decreased the copy number of the RABV N gene.And the copy number of RABV N gene was comparable to that of the control after the addition of nystatin was no significant difference.The results show the same between the N2 a cell and SH-SY5 Y and Vero cells.The above results indicated that RABV enters N2 a cells spend 2 hours.And RABV enters N2 a,SY5Y and Vero cells independently of the caveolin pathway but relies on clathrin-mediated,dynamin-and endosome low-p H mediated to enter cells.These results extended new insights into the mechanisms of rabies virus infection and provided novel targets for antiviral drug development.