Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endoso...Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.展开更多
基金supported by the National Key Research and Development Program of China(Grant No.216YFD0500402)Natural Science Foundation of China(Grants No.31272579 and 31472208)
文摘Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.
