Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus
Abstract Background Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III gras...
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BMC
2018-05-01
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| Series: | Virology Journal |
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| Online Access: | http://link.springer.com/article/10.1186/s12985-018-0993-8 |
| _version_ | 1811326618030833664 |
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| author | Hao Wang Weisha Liu Meng Sun Dubo Chen Lingbing Zeng Liqun Lu Jing Xie |
| author_facet | Hao Wang Weisha Liu Meng Sun Dubo Chen Lingbing Zeng Liqun Lu Jing Xie |
| author_sort | Hao Wang |
| collection | DOAJ |
| description | Abstract Background Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood. Methods Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR. Results Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-β-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore. Conclusions Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication. |
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| institution | Directory Open Access Journal |
| issn | 1743-422X |
| language | English |
| last_indexed | 2024-04-13T14:52:07Z |
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| spelling | doaj.art-017e2ce166174c83bf7ad99c2273da9b2022-12-22T02:42:33ZengBMCVirology Journal1743-422X2018-05-0115111010.1186/s12985-018-0993-8Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirusHao Wang0Weisha Liu1Meng Sun2Dubo Chen3Lingbing Zeng4Liqun Lu5Jing Xie6National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean UniversityNational Pathogen Collection Center for Aquatic Animals, Shanghai Ocean UniversityNational Pathogen Collection Center for Aquatic Animals, Shanghai Ocean UniversityDepartment of Laboratory Medicine, the frist affiliated hospital of Sun Yat-sen UniversityDivision of Fish Disease, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery SciencesNational Pathogen Collection Center for Aquatic Animals, Shanghai Ocean UniversityShanghai Engineering Research Center of Aquatic Product Processing & PreservationAbstract Background Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood. Methods Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR. Results Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-β-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore. Conclusions Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.http://link.springer.com/article/10.1186/s12985-018-0993-8Ctenopharyngodon idellusGCRV104Ammonium chlorideClathrinSedoreovirinae |
| spellingShingle | Hao Wang Weisha Liu Meng Sun Dubo Chen Lingbing Zeng Liqun Lu Jing Xie Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus Virology Journal Ctenopharyngodon idellus GCRV104 Ammonium chloride Clathrin Sedoreovirinae |
| title | Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus |
| title_full | Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus |
| title_fullStr | Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus |
| title_full_unstemmed | Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus |
| title_short | Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus |
| title_sort | inhibitor analysis revealed that clathrin mediated endocytosis is involed in cellular entry of type iii grass carp reovirus |
| topic | Ctenopharyngodon idellus GCRV104 Ammonium chloride Clathrin Sedoreovirinae |
| url | http://link.springer.com/article/10.1186/s12985-018-0993-8 |
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