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

Jie Gao,Xinyu Wang,Mingxin Zhao,Enhua Liu,Maolin Zhang,Ming Duan,Yidi Guo,Zhenhong Guan

doi:10.1186/s12985-019-1186-9

Abstract

BackgroundRabies 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.MethodsChemical 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.ResultsWe 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.ConclusionsOur results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.

Highlights

Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus
The results indicated that chlorpromazine and knockdown of clathrin heavy chains (CHC) reduced challenge virus standard (CVS)-11 infection, CVS-11 entry was not affected by nystatin or knockdown of caveolin-1
RABV entry is dependent on clathrin-mediated endocytic pathways Previous studies have shown that RABV endocytosis is dependent on clathrin

Summary

Introduction

Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. Rabies virus (RABV), a member of the genus Lyssavirus within the family Rhabdoviridae, which is a neurotropic pathogen, causes encephalomyelitis and high mortality in animals and humans. Most members of Rhabdoviridae family such as Vesicular stomatitis virus (VSV) [8], Australian bat lyssavirus (ABLV) [9], infectious hematopoietic necrosis virus (IHNV) [10] or Bovine ephemeral fever virus (BEFV) internalize host cells through clathrin-mediated endocytosis [11]. Using a recombinant VSV of which the endogenous glycoprotein was replaced with that of RABV (rVSV RABV G), previous studies have found that RABV internalized African green monkey kidney cell line (BS-C-1) [12] and peripheral neurons [13] through clathrin-mediated endocytic pathway by pharmacological perturbations or protein abundance, while G protein is the key factor to facilitate endocytosis. The mechanisms by which RABV enters cells are not well characterized

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