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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 w... Full description

Journal Title: Virology Journal 01 June 2019, Vol.16(1), pp.1-12
Main Author: Jie Gao
Other Authors: Xinyu Wang , Mingxin Zhao , Enhua Liu , Ming Duan , Zhenhong Guan , Yidi Guo , Maolin Zhang
Format: Electronic Article Electronic Article
Language: English
Subjects:
N2a
ID: E-ISSN: 1743-422X ; DOI: 10.1186/s12985-019-1186-9
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title: Entry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway
format: Article
creator:
  • Jie Gao
  • Xinyu Wang
  • Mingxin Zhao
  • Enhua Liu
  • Ming Duan
  • Zhenhong Guan
  • Yidi Guo
  • Maolin Zhang
subjects:
  • Rabv
  • N2a
  • Clathrin
  • Caveolin-1
  • Endocytosis
  • Medicine
  • Biology
ispartof: Virology Journal, 01 June 2019, Vol.16(1), pp.1-12
description: 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.
language: eng
source:
identifier: E-ISSN: 1743-422X ; DOI: 10.1186/s12985-019-1186-9
fulltext: fulltext_linktorsrc
issn:
  • 1743-422X
  • 1743422X
url: Link


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titleEntry of Challenge Virus Standard (CVS) -11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway
creatorJie Gao ; Xinyu Wang ; Mingxin Zhao ; Enhua Liu ; Ming Duan ; Zhenhong Guan ; Yidi Guo ; Maolin Zhang
ispartofVirology Journal, 01 June 2019, Vol.16(1), pp.1-12
identifierE-ISSN: 1743-422X ; DOI: 10.1186/s12985-019-1186-9
subjectRabv ; N2a ; Clathrin ; Caveolin-1 ; Endocytosis ; Medicine ; Biology
descriptionAbstract 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.
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description

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.

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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.

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