Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells
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Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-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-SY5Y cells was investigated. Immunofluorescence, TCID50 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.
KeywordsRab5 Rab7 rabies virus(RABV) endosomes colocalization
The study was supported by the National Key Research and Development Program of China (Grant No. 216YFD 0500402) and Natural Science Foundation of China (Grants No. 31272579 and 31472208).
WA, YYL, YDG, and XYW performed the experiments. MD and ZHG designed the study plan, performed image analysis. ZSL and MLZ performed statistical analysis and drafted the article. WA and YYL critically revised the article. All authors read and approved the final manuscript.
Compliance with Ethical Guidelines
The authors declare that they have no conflict of interest. The guidelines pertaining to care and welfare of laboratory animals were fully abided by the Animal Care and Use Committee of Jilin University.
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