石斑魚熱休克同源蛋白 70 與電壓依賴性陰離子通道蛋白 2 在 神經性壞死症病毒感染中的角色

Roles of grouper heat shock cognate protein 70 and voltage-dependent anion selective channel protein 2 in NNV infection in vitro

張瑞昕(Jui-Shin Chang)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/生命科學系/博士(2015年)

https://doi.org/10.6342/NTU.2015.00542

摘要

神經性壞死症病毒(nervous necrosis virus, NNV)是世界多種海水養殖魚的重要病 原,感染魚的高死亡率已造成許多海水養殖魚業嚴重損失。此病毒分類上屬於野田病毒科 (Nodaviridae),β 野田病毒屬(betanodavirus),為不具封套膜正二十面體病毒顆粒,具 二段正意單股核糖核酸(positive sense single strand RNA)。關於此病毒感染機制的相關文 獻仍有限,且尚未找到感染宿主之專一性受體蛋白質。在本論文第二章,利用病毒蛋白疊合 試驗(virus overlay protein binding assay, VOPBA),初步篩選出 GF-1 細胞膜系蛋白中能與 NNV 交互作用的蛋白質,其中包括石斑魚熱休克同源蛋白 70(grouper heat shock cognate protein 70, GHSC70)與石斑魚電壓依賴性陰離子通道蛋白 2(grouper voltage-dependent anion selective channel protein 2, GVDAC2)。將這兩種蛋白質基因解碼後,設計專一性小片 段干擾 RNA(Small interfering RNA, siRNA)分別抑制其基因表現,再感染 NNV,結果病 毒 RNA2 基因複製量明顯減少,說明這兩個蛋白對 NNV 感染或複製過程有影響 。經由免 疫沉澱實驗,發現 GHSC70 與 NNV 外鞘有交互作用,但 GVDAC2 則無。經螢光免疫染色 及流式細胞儀分析, GHSC70 可存在 GF-1 細胞膜上,且與吸附細胞後的 NNV 位置重疊。 最後以 GHSC70 抗血清預先處理 GF-1 細胞後再感染 NNV,可顯著降低細胞內病毒量。因 此證明,GHSC70 在 NNV 感染進入 GF-1 細胞過程中扮演著重要的角色,推測為 NNV 感染 宿主細胞的受體之一。論文第三章則探討 GVDAC2 在 NNV 感染過程中扮演角色。NNV 感 染並不影響 GF-1 細胞中 GVDAC2 基因表現量的大幅改變。細胞免疫染色結果顯示, GVDAC2 和 NNV RNA 聚合酶皆座落於粒腺體上,但免疫沉澱結果顯示此兩蛋白質並無直 接交互作用。以 siRNA 抑制細胞 GVDAC2 表現後,細胞中 ATP 量顯著下降;NNV 在感染 GVDAC2 表現量受到抑制的 GF-1 細胞後,NNV 誘發的細胞凋亡時程會延後。因此認為, GVDAC2 在 NNV 感染細胞過程中,對維持細胞中 ATP 量以及感染後期病毒誘發細胞凋亡 的時程具有重要性。

關鍵字

神經壞死症病毒 ； 石斑魚鰭細胞株 GF-1 ； 石斑魚熱休克同源蛋白質70 ； 石斑魚電壓性陰離子通道蛋白質2 ； 病毒受體 ； 細胞凋亡

並列摘要

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.

並列關鍵字

Nervous necrosis virus (NNV) ； grouper fin cell line GF-1 ； grouper heat shock cognate protein 70 (GHSC70) ； grouper voltage-dependent anion selective channel protein 2 (GVDAC2) ； viral receptor ； apoptosis

參考文獻

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