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Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus

文献类型： 外文期刊

作者： Wang, Hao 1 ; Liu, Weisha 1 ; Sun, Meng 1 ; Chen, Dubo 4 ; Zeng, Lingbing 5 ; Lu, Liqun 1 ; Xie, Jing 6 ;

作者机构： 1.Shanghai Ocean Univ, Natl Pathogen Collect Ctr Aquat Anim, Shanghai, Peoples R China

2.Minist Agr & Rural Affairs, Key Lab Freshwater Aquat Genet Resources, Shanghai, Peoples R China

3.Shanghai Ocean Univ, Natl Demonstrat Ctr Expt Fisheries Sci Educ, Shanghai, Peoples R China

4.Sun Yat Sen Univ, Dept Lab Med, Affiliated Hosp 1, Guangzhou, Guangdong, Peoples R China

5.Chinese Acad Fishery Sci, Yangtze River Fisheries Res Inst, Div Fish Dis, Wuhan, Hubei, Peoples R China

6.Shanghai Engn Res Ctr Aquat Prod Proc & Preservat, Shanghai, Peoples R China

关键词： Ctenopharyngodon idellus; GCRV104; Ammonium chloride; Clathrin; Sedoreovirinae

期刊名称：VIROLOGY JOURNAL （ 影响因子：4.099； 五年影响因子：3.719 ）

ISSN： 1743-422X

年卷期： 2018 年 15 卷

页码：

收录情况： SCI

摘要： 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-beta-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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