白點症病毒藉由PI3K-Akt-mTOR訊息路徑調控的瓦氏效應以平衡宿主細胞之氧化壓力

White spot syndrome virus infection promotes the PI3K-Akt-mTOR pathway-regulated Warburg effect to reduce oxidative stress in Penaeus vannamei

陳顗同(I-Tung Chen)

指導教授 ： 羅竹芳

共同指導教授 ： 郭光雄

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.2015.00889

摘要

本研究我們探討蝦白點症病毒如何改變宿主細胞的代謝反應，維持細胞內氧化還原狀態的平衡，以中和於感染初期免疫細胞於病毒感染初期所誘發的活性氧物質。在白點症病毒感染初期30分鐘至2小時內，蝦血球細胞內活性氧物質的含量大量累積，造成細胞內的氧化傷害。本實驗分析氧化壓力指標 NADPH/NADP+ 及 GSH/GSSG 之比值以檢視細胞內之氧化還原狀態，結果顯示蝦類細胞中氧化還原指標於感染初期2小時內血球細胞處於不平衡的氧化狀態。隨著感染時序往後，細胞內強還原劑如 NADPH 及 GSH 之相對濃度增加進而平衡細胞內的氧化壓力。我們亦發現蝦血清中乳酸濃度累積以及NADH/NAD+ 比值於病毒DNA複製時期持續上升，顯示白點症病毒感染造成瓦氏效應的發生，此舉有助於中和感染初期細胞內大量的活性氧物質。藉由施打PI3K-Akt-mTOR訊息路徑抑制劑證明，白點症病毒所引起的瓦氏效應與細胞內氧化還原狀態平衡的關聯性。代謝體學分析證明了白點症病毒啟動了有氧醣解的代謝改變，刺激反應進行，細胞大量且快速的消耗醣類來源，然而磷酸烯醇丙酮酸 (PEP) 於感染細胞中產量逐漸累積，且下游產物丙酮酸 (pyruvate) 產量減少，中間代謝酵素丙酮酸激酶 (pyruvate kinase) 的催化活性受到抑制，故導致磷酸烯醇丙酮酸的累積，及上游產物的推積，致使代謝路徑進而轉向五碳醣磷酸途徑，在瓦氏效應下，細胞於短時間內可獲得大量的能量 (NADH) 以及還原物質來源 (NADPH)。抗氧化物質NADPH之合成上升，以消弭免疫細胞產生之活性氧化物。因此病毒感染細胞內氧化還原狀態趨向平衡，有助於於白點症病毒基因之功能表現與複製行為。

關鍵字

活性氧物質 ； 白點症病毒 ； 氧化還原平衡 ； 瓦氏效應 ； 系統生物學 ； 代謝體學

並列摘要

Here we investigate how white spot syndrome virus (WSSV) alters its host’s metabolism to restore host redox balance and counter the immediate increase in ROS production that occurs after WSSV infection. Intracellular ROS levels were significantly increased in hemocytes collected from WSSV-infected shrimp within the first 30 min ~ 2 h post infection. Measurement of the NADPH/NADP+ and GSH/GSSG ratios revealed that after a significant initial imbalance toward the oxidized forms at 2 hpi, redox equilibrium was subsequently restored. Together with high levels of lactic acid production and an elevated NADH/NAD+ ratio, these changes suggest that WSSV induces aerobic glycolysis to counteract the high levels of ROS production that were triggered in response to viral infection. Suppression of the virus-induced Warburg effect by chemical inhibitor of PI3K-Akt-mTOR singling pathway cause the sustained production in intracellular ROS. A metabolomics analysis confirmed that glycolysis activity in shrimp was up-regulated after WSSV infection. However, pyruvate kinase activity was inhibited by WSSV, and accumulation of upstream metabolites caused glucose metabolism to be redirected into the pentose phosphate pathway and the serine and glycine biosynthesis pathway. Here we show that the initially high levels of ROS trigger by WSSV is combated by PI3k-Akt-mTOR regulated Warburg effect. Changes induced by WSSV infection cause re-routing of the host’s glycolytic metabolism into subsidiary pathways and increases the availability of raw materials that are essential for WSSV gene expression and replication.

並列關鍵字

ROS ； WSSV ； PI3K-Akt-mTOR ； Redox balance ； Warburg effect ； Systems biology ； metabolomics.

參考文獻

1. Akaike T. (2001) Role of free radicals in viral pathogenesis and mutation. Rev Med Virol. 11:87-101.

2. Akaike T, Noguchi Y, Ijiri S, Setoguchi K, Suga M, Zheng YM, Dietzschold B, Maeda H. (1996) Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci U S A. 93:2448-2453.

3. Alic N, Higgins VJ, Dawes IW. (2001) Identification of a Saccharomyces cerevisiae gene that is required for G1 arrest in response to the lipid oxidation product linoleic acid hydroperoxide. Mol Biol Cell. 12:1801-1810.

4. Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS, Thomas CJ, Vander Heiden MG, Cantley LC. (2011) Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science. 334:1278-1283.

5. Archer SL, Huang JMC, Hampl V, Nelson DP, Shultz PJ, Weir EK. (1994) Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K-channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA. 91:7583-7587.