自噬作用為細胞內維持恆定的代謝機制，可將老舊或受損胞器分解回收，以提供能量合成新的胞器或其他用途。而當細胞面臨壓力或病原入侵時也會誘導發生。實驗室先前研究證實神經壞死病毒(RGNNV)感染石斑魚鰭細胞株(GF-1)會產生由活性氧分子(O2-, H2O2)所介導的自噬作用。因此本研究想要探討在病毒感染下所誘導之活性氧分子如何調節自噬作用。首先由蛋白質表現分析結果顯示，病毒感染使自噬作用上游分子mTOR活性上升，並由AMPK及Akt所調控。若添加抗氧化劑DPI(Diphenylene iodonium)抑制O2-生成能減少mTOR活性，而添加GSH(Glutathione)促H2O2代謝則無法降低mTOR活性。接著進一步由穩定表現mRFP-LC3細胞株及MDC染色觀察病毒感染所誘導之自噬作用，結果發現，抑制O2-生成或促進H2O2代謝均能降低自噬作用，其中又以抑制O2-能降低的效果最為明顯，且由蛋白質表現分析也觀察到抑制O2-能降低Class III PI3K(Vps34)的表現量。在病毒效價及細胞存活方面，抑制O2-能降低病毒效價並提高細胞存活。 此外抗氧化轉錄因子Nrf2，近來文獻顯示會和自噬作用中的sequestosome 1/p62 (SQSTM1/p62)蛋白具有交互作用以調節細胞中氧化壓力。在我們系統中亦發現，若降低病毒感染所誘導的活性氧分子，Nrf2及p62的表現會減少。若進一步利用Nrf 2抑制劑RA(Retinoic Acid)或p62 siRNA，顯示 Nrf2和p62在RGNNV感染下有交互作用。總結以上結果可知，O2-為病毒感染所誘導的主要活性氧分子，並藉由調節mTOR活性及Class III PI3K(Vps34)之表現，參與病毒誘發的自噬作用及細胞死亡，另外也調節Nrf2及抗氧化酵素的表現。

Autophagy is a cellular metabolic process to degrade and recycle the older or damaged organelle that autophagic also induces under the stress or virus infection. In this study, we found that (1) antioxidant DPI (for blocking superoxide production) treatment with RGNNV-infected GF-1 cells can block mTOR activity, but did not blocked by GSH (for reducing hydrogen oxide production) treatment; (2) with MDC staining in mRFP-LC3 cells, we found that antioxidant DPI treatment can reduce O2- production and that can significantly reduce virus-induced autophagy and enhance cell viability; (3) we found that Class III PI3K(Vps34) also participates in virus-induced autophagy by reactive oxygen species. Finally, we have found that antioxidant transcription factor Nrf2 can interact with p62 by using specific inhibitor retinoic acid or p62 siRNA treatment in GF-1 cells with RGNNV infection. Taken together our results suggest that RGNNV-induced ROS (O2-) signals can regulate the autophagic process for enhancing viral replication and host cell death. Then, the autophagic process is regulated by mTOR and class III PI3K signals. Then, the mTOR signal is regulated by either AKT, or AMPK activities. Our finding provided insight into RNA virus molecular mechanism.

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