白點症病毒(White spot syndrome virus, WSSV)造成養蝦產業經濟衝擊及嚴重損失，因而其致病機轉為當前重要研究課題。先前本實驗室觀測少數代謝物佐以label-free蛋白質體學系統，發現WSSV感染後會誘使宿主代謝路徑改變，在基因體複製階段時，參與醣解代謝路徑中大部份蛋白質皆呈現表現量上升之趨勢，並伴隨乳酸大量生成，類似於癌症細胞當中的瓦氏效應(Warburg effect)。為了進一步釐清瓦氏效應與WSSV致病機轉之關聯性，本研究利用高靈敏LC-ESI-MS代謝體分析平台，全面性觀測WSSV感染後蝦血細胞整體代謝變化，發現醣解路徑、五碳醣磷酸路徑、核苷酸生合成及檸檬酸循環等中間代謝產生會因WSSV不同感染時程而有規律變化。研究結果顯示，mTOR訊息傳遞路徑(mTOR signaling pathway)可因WSSV感染而活化，可能為驅使代謝路徑改變之作用機制。探討後發現mTOR上游作用因子PI3K/Akt經抑制劑處理後，病毒基因表現及病毒顆粒累積量皆有顯著下降，而利用基因沉默主要mTORC1的活化因子Rheb卻無法有效的抑制病毒增殖，因此推測或許有其他路徑驅使下游mTOR活化，因此轉而針對mTORC1與非主流作用之mTORC2以抑制物Torin1進行共抑制。結果發現若共抑制mTORC1 與mTORC2後，除了WSSV所產生之瓦氏效應消失外，病毒基因表現及病毒顆粒累積量也顯著下降，因此推測mTOR訊息傳遞路徑在WSSV誘發之瓦氏效應中扮演重要角色。

WSSV (White Spot Syndrome Virus) has emerged globally and caused huge economic damage to the shrimp farming industry. However, the pathogenesis of WSSV remains unclear. Our previous study found that, the Warburg effect, a common metabolic change in cancer cells, was present in shrimp hemocytes in the genome replication stage of WSSV infection. To further clarify the mechanism of WSSV-induced Warburg effect, we used the liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) approach to investigate the metabolic alteration during WSSV infection. We found that the metabolic alteration of glycolysis, the pentose phosphate pathway, and even the citric acid cycle is happened during WSSV infection. Besides, after WSSV infection, the mTOR activity was up-regulated, suggesting that mTOR signaling pathway may be triggered the metabolic changes. We further evaluate the relationship between mTOR pathway and WSSV-induced Warburg effect. The results showed that the inhibition of mTOR upstream factor, PI3K/Akt, led to the decrease of WSSV gene expression and viral copy numbers. Subsequently, the silencing of mTORC1 activator, Rheb, has no effect to WSSV pathogenesis, suggesting that some alternative factors may be triggered, such as mTORC2. By using Torin 1, an inhibitor of mTORC1 and mTORC2, we found that this treatment was significantly decreased the WSSV gene expression, viral copy numbers, and the WSSV-induced Warburg effect. Taken together, this study demonstrated that mTOR signaling pathway play a crucial role in WSSV induced Warburg effect.

Aspuria PJ, Tamanoi F. (2004) The Rheb family of GTP-binding proteins. Cell Signal. 16: 1105–1112.
Buerger C, DeVries B, Stambolic V. (2006) Localization of Rheb to the endomembrane is critical for its signaling function. Biochemical and Biophysical Research Communications. 344: 869-880.
Bishop NA, Guarente L. (2007) Genetic links between diet and lifespan: shared mechanisms from yeast to humans. Nature Reviews Genetics. 8(11): 835-44.
Bjedov I, Toivonen JM, Kerr F, Slack C, Jacobson J, Foley A, Partridge L. (2010) Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metabolism. 11: 35-46.
Buchkovich NJ, Yu Y, Zampieri CA, Alwine JC. (2008) The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K-Akt-mTOR signalling pathway. Nature Reviews Microbiology. 6: 266-275.
Busk M, Horsman MR, Kristjansen PE, van der Kogel AJ, Bussink J, Overgaard J. (2008) Aerobic glycolysis in cancers: implications for the usability of oxygen-responsive genes and fluorodeoxyglucose-PET as markers of tissue hypoxia. International Journal of Cancer. 122: 2726-2734.
Chen IT, Aoki T, Huang YT, Hirono I, Chen TC, Huang JY, Chang GD, Lo CF, Wang HC. (2011) White spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection. Journal of Virology. 85: 12919-28.
Clippinger AJ, Maguire TG, Alwine JC. (2011) Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation. Journal of Virology. 85: 9369-76.
Clippinger AJ, Maguire TG, Alwine JC. (2011) The Changing Role of mTOR Kinase in the Maintenance of Protein Synthesis during Human
Cytomegalovirus Infection. Journal of Virology. 85: 3930-9.
Drenan RM, Liu X, Bertram PG, Zheng XF. (2004) FKBP12 – rapamycin - associated protein or mammalian target of rapamycin (FRAP/mTOR) localization in the endoplasmic reticulum and the Golgi apparatus. Journal of Biological Chemistry. 279: 772-778.
Düvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. (2010) Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Molecular Cell. 39: 171-83.
Dunn EF, Connor JH. (2011) Dominant inhibition of Akt/protein kinase B signaling by the matrix protein of a negative-strand RNA virus. Journal of Virology. 85: 422-431.
Diamond DL, Syder AJ, Jacobs JM, Sorensen CM, Walters KA, Proll SC, McDermott JE, Gritsenko MA, Zhang Q, Zhao R, Metz TO, Camp DG 2nd, Waters KM, Smith RD, Rice CM, Katze MG. (2010) Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLOS Pathogens. 6:e1000719.
DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. (2008) The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metabolism. 7: 11-20.
Delgado T, Carroll PA, Punjabi AS, Margineantu D, Hockenbery DM. (2010) Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells. Proceedings of the National Academy of Sciences. 107: 10696-10701.
Dang CV. (2012) Links between metabolism and cancer. Genes & Development. 26: 877-890
Escobedo-Bonilla CM, Alday-Sanz V, Wille M, Sorgeloos P, Pensaert MB and Nauwynck HJ. (2008) A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome Virus. Journal of Fish Diseases. 31:1-18.
Guarente L. (2000) Sir2 links chromatin silencing, metabolism, and aging. Genes & Development. 14: 1021-6
Gaoa N, Zhangc Z, Jianga BH, Shi X. (2003) Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer. Biochemical and Biophysical Research Communications. 310: 1124-1132
Hansen M, Taubert S, Crawford D, Libina N, Lee SJ, Kenyon C. (2007) Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell. 6: 95-110.
Hollenbaugh JA, Munger J, Kim B. (2011) Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis. Virology. 415: 153-159.
Hay N , Sonenberg N. (2004) Upstream and downstream of mTOR. Genes & Development. 18: 1926-1945.
Jia K, Chen D, Riddle DL (2004) The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development. 131: 3897-3906.
Kang ST, Leu JH, Wang HC, Chen LL, Kou GH, Lo CF. (2009) Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes. Virology. 387: 353-63.
Kerry DF, Bert LS. (2009) Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochim Biophys Acta. 1789: 518-528.
Kim HS, Xiao C, Wang RH. (2010) Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis. Cell Metabolism. 12: 224-36.
Kim JW, Tchernyshyov I, Semenza GL, Dang CV. (2006) HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia. ELSEVIER INC. 3: 177-185.
King A, Selak MA , Gottlieb E. (2006) Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer. Oncogene. 25: 4675-4682.
Kudchodkar SB, Yu Y, Maguire TG, Alwine JC. (2006) Humancytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes. Proceedings of the National Academy of Sciences. 103: 14182-14187.
Lo CF, Leu JH, Ho CH, Chen CH, Peng SE, Chen YT, Chou CM, Yeh PY, Huang, CJ, Chou HY, Wang CH, Kou GH. (1996) Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimp using polymerase chain reaction. Disease of Aquatic Organisms. 25: 133-141.
Leu JH, Chang CC, Wu JL, Hsu CW, Hirono I, Aoki T, Juan HF, Lo CF, Kou GH, Huang HC. (2007) Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon. BMC Genomics. 1186: 1471-2164.
Laplante M and Sabatini DM. (2009) mTOR signaling at a glance. Journal of Cell Science. 122: 3589-3594.
Munger J, Bajad SU, Coller HA, Shenk T, Rabinowitz JD. (2006) Dynamics of the cellular metabolome during human cytomegalovirus infection. PLOS Pathogens. 2: e132.
Munger J, Bennett BD, Parikh A, Feng XJ, McArdle J, et al. (2008) Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nature Biotechnology. 26: 1179-1186.
Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T. (2008) Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Cell Host Microbe. 3: 253-262.
Moorman NJ, Shenk T. (2010) Rapamycin-resistant mTORC1 kinase activity is required for herpesvirus replication. Journal of Virology. 84: 5260-5269.
Nardella C, Carracedo A, Alimonti A, Robin M, Hobbs, John G, Clohessy. (2009) Differential requirement of mTOR in postmitotic tissues and tumorigenesis. Science Signaling. 27; 2(55):ra2.
Ponisovskiy MR. (2010) Cancer Metabolism and the Warburg Effect As Anabolic Process Outcomes of Oncogene Operation. Critical reviews in eukaryotic gene expression. 20: 325-339.
Sato T, Umetsu A, Tamanoi F. (2008) Characterization of the Rheb-mTOR Signaling Pathway in Mammalian Cells: Constitutive Active Mutants of Rheb and mTOR. Methods in Enzymology. 438: 307-320.
Shim H, Dolde C, Lewis BC, Wu CS, Dang G, Jungmann RA, Dalla-Favera R, Dang CV. (1997) c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proceedings of the National Academy of Sciences. 94: 6658-6663.
Sun Q, Chen X, Ma J, Peng H, Wang F, Zha X, Wang Y, Jing Y, Yang H, Chen R, Chang L, Zhang Y, Goto J, Onda H, Chen T, Wang MR, Lu Y, You H, Kwiatkowski D, Zhang H. (2011) Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proceedings of the National Academy of Sciences. 108: 4129-34.
Vander Heiden MG, Cantley LC, Thompson CB. (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 324: 1029-1033.
Vastag L, Koyuncu E, Grady SL, Shenk TE, Rabinowitz JD. (2011) Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism. PLOS Pathogens. 7: e1002124.
Wang HC, Wang HC, Leu JH, Kou GH, Wang AH, Lo CF. (2007) Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Developmental Comparative Immunology. 31(7): 672-86.
Warburg O. (1956) On respiratory impairment in cancer cells. Science. 124: 269-270.
Whiteman EL, Cho H, Birnbaum MJ. (2002) Role of Akt/protein kinase B in metabolism. Trends in Endocrinology and Metabolism. 13: 444-451.
Wullschleger S, Loewith R, Hall M.N. (2006) TOR signaling in growth and metabolism. Cell. 124: 471-484.
Yamagata K, Sanders LK, Kaufmann WE, Yee W, Barnes CA, Nathans D, Worley PF. (1994) rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein. The Journal of Biological Chemistry. 269: 16333-16339.
Zhong L, D’Urso A, Toiber D, Sebastian C, Henry RE, Vadysirisack DD, Guimaraes A, Marinelli B, Wikstrom JD, Nir T. (2010) The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell. 140: 280-293.