神經壞死病毒外殼蛋白與多聚腺苷酸結合蛋白於石斑魚腦細胞之交互作用

Abstract

神經壞死病毒(nervous necrosis virus, NNV)為一種對魚類致死率極高之病毒，其主要之攻擊目標為神經系統，包含腦、脊髓與視網膜等，終致感染魚隻沉底死亡。此病毒挾其高致死率令全球養殖業蒙受巨大的經濟損失。為瞭解神經壞死病毒之感染機轉，本實驗室以大腸桿菌表現神經壞死病毒殼蛋白，此殼蛋白會自行組裝為類病毒顆粒(virus-like particle, VLP)。以此 VLP 製備親和性管柱，用以純化點帶石斑魚腦(grouper brain, GB)細胞之結合蛋白，此結合蛋白經 LC/MS/MS 鑑定分析，結果顯示 polyadenylate binding protein(PABP)與類病毒顆粒具有非常高的親和性。PABP 為細胞正規轉譯機制中，強化轉譯效率之重要蛋白。部分病毒以此為標的來抑制宿主細胞轉譯能力，而 NNV的 RNA基因體與訊息 RNA 並不具有 3’端多聚腺苷尾。因此殼蛋白與 PABP 間強大的親和性，引起我們高度的興趣。點帶石斑魚的 PABP 基因編碼 633 個胺基酸分子量推估為 69, 360 dalton，N 端含有 4 個 RNA recognition motifs (RRM)，C 端含有一 C- terminal domain，其間為 proline-rich linker。此基因相當保守與人類細胞之 PABP 於核酸序列有 94.0%之相似性，於胺基酸序列則有 99.8%相似。為了瞭解 NNV 感染 GB 細胞的轉譯調控，首先以 puromycin 標示法利用免疫細胞化學染色分析新合成蛋白，可觀察到感染後 12 小時呈現幾乎全面的轉譯關閉。 且此細胞轉譯關閉與 PABP 的入核、降解和 NNV coat protein 的出現與入核具有密切的重 疊相關性。接著以 rNNV CP 單一表現載體轉染至 GB 細胞，發現 rNNV CP 之表現的確導 致細胞轉譯關閉與PABP之入核與降解。而透過免疫共沉澱可進一步證明NNV CP與PABP 確實存在交互作用，更以 Far-Western 確認兩者間的結合區位於 NNV coat protein 之 1-150 aa 與 PABP 的 370-538 aa。在病毒感染後期，我們觀察到 PABP 逐漸降解。此病毒感染後 的 PABP 降解現象以 MG132 抑制和 anti-ubiquitin 共沉澱分析證實是經由 26S-proteasome 進 行降解。總結，NNV 透過 coat protein 與 PABP 的交互作用導致 PABP 的核轉置與降解， 進而劫持宿主 GB 細胞的轉譯。

Nervous necrosis virus (NNV) belonging to Betanodavirus of the Nodaviridae family is the caused agent of viral nervous necrosis (VNN) disease in larvae and juvenile of marine fish. The devastating infection led to the huge economic damage to aquaculture industry. However, the hijacking mechanism of NNV on its host is less understood. We found that the host translation had been hijacked and shutoff after NNV infection in grouper brain(GB) cells by using puromycin labeling SUnSET detection. The SUnSET assay showed that the high moi NNV infection result in GB cell translation shutdown 6 hpi and almost shutoff 12 hpi. The NNV genomes and mRNA contain a cap structure on their 5’-terminal as that of cellular canonical mRNA, but no poly(A)- tail on their 3’-terminal. Furthermore, the result of LC/MS/MS identified associated proteins by recombinant NNV coat protein affinity column showed that the polyadenylate binding protein (PABP) is highly associated with NNV coat protein. Therefore, we wonder whether PABP is the target for NNV to shutoff host translation. The orange-spotted grouper PABP gene encodes 633 aa open reading frame with 69, 360 dalton in molecular weight and contains four RNA recognition motifs, one proline-rich linker and one C-terminal domain. The PABP is an important factor for canonical translation. We found that the PABP of orange-spotted grouper is highly conservative with that of human in nucleotide and amino acid sequence with 94.0% and 99.8% similarity, respectively. From immunocytochemistry result, we observed that the appearance and nucleus localization of NNV coat protein is highly correlative to the PABP nucleus relocalization and degradation as well as host translation shutoff in NNV-infected GB cells. The nucleus relocalization and degradation of PABP were further proved by the only expressed recombinant NNV coat protein in GB cells. Next, the coimmunoprecipitation experiment confirmed the interaction between NNV coat protein and PABP, and the interaction regions were further defined to NNV coat protein 1-150 aa and PABP 370-538 aa region by Far-Western blot. In the late stage of infection, the PABP is gradually degraded. The inhibition of PABP degradation by the treatment of MG132, and the finding of truncated form PABP in the anti-ubiquitin immunoprecipitation suggested that the PABP degradation induced by NNV infection is through ubiquitin-26S proteasome pathway. Our results supported that the NNV coat protein hijack PABP to relocalize into nucleus and mediated its late infection stage degradation which cause the host translation shutoff eventually.