白點症病毒蛋白質激酶透過與宿主鐵蛋白交互作用影響宿主鐵衡定之分析

鐵對於宿主與病原體皆為生存必需之因子，宿主亦可利用限制細胞內自由鐵的機制達到防禦目的，而病原體也常有其因應策略。在本實驗中，利用酵母菌雙雜交系統、pull-down 分析或石英晶體微天平分析證實，白點症病毒蛋白質激酶 PK1 與 WSSV189 及宿主鐵蛋白三者之間具有交互作用。由於已知 pk1 為白點症病毒之必需基因，鐵蛋白則為細胞內最主要的儲鐵蛋白質，故針對 PK1 與鐵蛋白間之作用做進一步探討。以 pull-down 分析及石英晶體微天平分析證實，PK1 除可與攜有鐵的鐵蛋白作用外，亦可與脫鐵蛋白產生交互作用。而利用細胞外的鐵離子結合試驗得知，PK1 雖無法獨力促使鐵離子由鐵蛋白中釋出，但卻可抑制脫鐵蛋白與亞鐵離子結合。當以轉染作用於昆蟲細胞 Sf9 中表現 PK1 時，其細胞內 labile iron pool (LIP) 會高於對照組，甚至達到以檸檬酸銨鐵處理細胞時 LIP 所上升的程度，顯示 PK1 具有可抑制脫鐵蛋白穩定細胞 LIP 程度的特性。此外，利用免疫沈澱法與石墨爐式原子吸收光譜儀分析時可發現，當蝦體受白點症病毒感染 48 小時後，體內每單位總鐵蛋白中的鐵含量較對照組下降約達 83.4%，顯示白點症病毒感染確實會影響蝦體內鐵蛋白的攜鐵含量。而干擾宿主對自由鐵的限制，對於白點症病毒而言是將有利的，因為鐵對核醣核苷酸還原酶活性具有關鍵角色，而白點症病毒亦可表現核醣核苷酸還原酶，此酵素對於病毒可於宿主細胞內快速增殖十分重要。依據本研究所獲得之結果推測，白點症病毒有可能藉由 PK1 抑制宿主鐵蛋白對自由鐵的限制，以取得足量的鐵，確保病毒核醣核苷酸還原酶的活性、維持病毒有效率地增殖。而此結果亦可能是病毒蛋白質透過與宿主鐵蛋白直接產生交互作用、影響其功能，以達到干擾宿主鐵衡定目的相關研究的首例。

關鍵字

白點症病毒；蛋白質激酶；鐵蛋白；鐵衡定

並列摘要

Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. The host iron withholding mechanism is considered part of innate immunity that can restrict iron availability to pathogens. However, pathogens demonstrate strategies for overcoming this obstacle. In this study, we used a yeast two-hybrid system and a pull-down assay, and introduced a 27-MHz quartz crystal microbalance (QCM) instrument to verify the interactions between the white spot syndrome virus (WSSV) PK1, WSSV189 and the host ferritin. Because pk1 is an essential gene for WSSV and ferritin is the major iron storage protein in cells, the interaction between PK1 and ferritin was examined closely in this study. In addition to the iron-containing ferritin, PK1 also interacts with apoferritin. These interactions can be confirmed using the pull-down assay and QCM instrument analysis. Although PK1 cannot promote iron ions released from ferritin, the results of the iron ion-binding assays revealed that the ferrous ion-binding activity of apoferritin can be blocked by PK1. Furthermore, when the PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly, even in the cells that were not treated with ferric ammonium citrate (FAC). This suggests that PK1 can affect apoferritin in stabilizing the cellular LIP levels. Moreover, using immunoprecipitation and graphite furnace atomic absorption spectrophotometer (AAS) detection, we discovered that the level of iron ions bound by ferritin decreased 84% after 48 h post-WSSV infection. Reduction of the iron sequestered by ferritin is a benefit to WSSV because iron plays a critical role in the ribonucleotide reductase (RR) activity. The WSSV genome has 2 RR genes that encode large and small subunits, respectively, and form a functional holoenzyme that enables WSSV to replicate in a nondividing cell. Because WSSV tends to require iron to enhance RR activity, we speculate that WSSV may inhibit the free iron sequestered by the host ferritin through the PK1 interactions with ferritin to ensure the activity of WSSV RR and maintain efficient proliferation in shrimp. This may be the first report on how the viral protein disrupts the function of ferritin through direct protein-protein interaction and affects the host iron homeostasis.

並列關鍵字

white spot syndrome virus ； WSSV ； protein kinase ； ferritin ； iron homeostasis

參考文獻

Andrews, S.C., 1998. Iron storage in bacteria. Adv. Microb. Physiol. 40, 281-351.

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Bevilacqua, M.A., Faniello, M.C., Quaresima, B., Tiano, M.T., Giuliano, P., Feliciello, A., Avvedimento, V.E., Cimino, F., Costanzo, F., 1997. A common mechanism underlying the E1A repression and the cAMP stimulation of the H ferritin transcription. J. Biol. Chem. 272, 20736-20741.

Bischoff, D.S., Slavicek, J.M., 1994. Identification and characterization of a protein kinase gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. J. Virol. 68, 1728-1736.

Burma, S., Mukherjee, B., Jain, A., Habib, S., Hasnain, S,E., 1994. An unusual 30-kDa protein binding to the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. J. Biol. Chem. 269, 2750-2757.

國際替代計量

白點症病毒蛋白質激酶透過與宿主鐵蛋白交互作用影響宿主鐵衡定之分析

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