Roles of grouper heat shock cognate protein 70 and voltage-dependent anion selective channel protein 2 in NNV infection in vitro
| Autor: | Jui-Shin Chang, 張瑞昕 |
|---|---|
| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish, and has affected more than 40 fish species. NNV belongs to the betanodavirus of Nodaviridae and is a non- enveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, the knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. In Chapter 2, we using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat-shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were presumably to be NNV receptor protein candidates. We cloned, sequenced, and expressed the genes of GHSC70 and GVDAC2 in Escherichia coli for anti-serum preparation. The expression knockdown of GHSC70 and GVDAC2 genes with specific short interfering RNA (siRNA) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. After an immuno- precipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 level after 1 h of adsorption to GF-1 cells, which was significantly lower in the cells pretreated with the GHSC70 antiserum than in non-treated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functions as NNV receptor or co-receptor protein. In Chapter 3, we investigated its role in the NNV infection. NNV infection did not considerably affect GVDAC2 gene expression. After performing immunostaining, we detected GVDAC2 at the mitochondrial membrane and GVDAC2 was colocalized with NNV-RNA-dependent RNA polymerase. However, these 2 proteins did not interact with each other in immunoprecipitation assay. The cellular ATP level in GVDAC2- downregulated cells was lower than that in control cells, and NNV-induced apoptosis was delayed in GVDAC2-siRNA-transfected cells. Therefore, we suggest that GVDAC2 is required for NNV infection for maintaining the cellular ATP level and had positive impact on virus-induced apoptosis. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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