Entry of Challenge Virus Standard (CVS)-11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway

Abstract

Background: Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. Methods: Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. Results: We firstly examined the endocytosis pathway of the challenge virus standard (CVS)-11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MβCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. Conclusions: Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.