Host Cell Entry Mechanism Of Human Metapneumovirus

Human metapneumovirus(h MPV)is an important pathogen causing infants and young children's acute respiratory infections(ARI).h MPV is a member of the Paramyxoviridae family.Although the studies suggest that paramyxoviruses initiate fusion at the plasma membrane,the entry mechanism for h MPV is largely uncharacterized.Here we sought to determine whether h MPV initiates fusion at the plasma membrane or following internalization.To study the h MPV entry process,human metapneumovirus were labeled with fusion protein antibody and the core protein antibody respectively.Bes ides we used an R18-dequenching fus ion assay,and developed a quantitative,fluorescence microscopy assay to visualize the cellular s ite of h MPV fusion.We found that h MPV particles are endocytic into Vero E6 cells.Then we studied the role of lipid rafts in endocytosis process of h MPV.Confocal microscope was employed to detect colocalization of h MPV F protein and a lipid raft marker.The results showed that colocalization was increased along with the viral infection duration and h MPV particles transferred to perinuclear region accompany with lipid raft.When specific lipid raft inhibitors such as methyl-? cyclodextrin and nystatin were used,the h MPV viral titer decreased dramatically.With the replenishment of exogenous cholesterol,h MPV reversed quickly.These data suggest that lipid raft plays an important role in h MPV endocytosis.PART ? The role of endocytosis in h MPV entryObjective: To study whether h MPV enter Vero E6 cells by endocytosisMethods: human metapneumovirus were labeled with fusion protein antibody and the core protein antibody respectively.The fusion protein(F protein)located in the envelope of the virus and the core protein(N protein)located in the nucleus.So we can observe whether there is a complete virus particles into cells.Bes ides we used a dequenching fus ion assay,and developed a quantitative,fluorescence microscopy assay to visualize the cellular site of h MPV fusion.Results: After labeling with fluorescent antibody.We found that F protein and N protein co-located at different time and was gradually increased with the incubation.R18 fusion experiments showed that red fluorescence appeared in Vero E6 cells.This results indicated that h MPV membrane and cell membrane fused intracellular.In the Di OC / R18 fus ion experiment,a yellow fluorescent spot was observed in the cytoplasm.This results further confirmed that the human metapneumovirus enters the cell and fuses in the cytoplasm.Conclusion: h MPV particles enter into Vero E6 cells by endocytosis.PART ? The Role of Lipid Rafts in Cell Entry of Human MetapneumovirusObjective: To study whether lipid rafts play an important role in endocytosis process in 16 HBE cells.Methods: We used fluorescent dyes to observe the relationship between lipid raft and h MPV in the process of h MPV entry.Besides,methyl-?-cyclodextrin and nystatin were used to disrupt cell lipids.At last,water soluble cholesterol was added to the culture system to study whether cholesterol could restore h MPV infection.Results: In this experiment,we found that the co-localization of CTB and h MPV increased gradually at 30/60/120 min.Only little co-locating points were found at 0 min,and some co-locating points were concentrated on the cell membrane at 30 min.By analyzing the results of 60/120 min,most of these co-locating points appear at the s ite away from the cell membrane.When specific lipid raft inhibitors such as methyl-? cyclodextrin and nystatin were used,the h MPV viral titer decreased dramatically.With the replenishment of exogenous cholesterol,h MPV reversed quickly.Conclusion: Our study illustrates lipid rafts play an important role in h MPV cell entry in human bronchial epithelial cell.