Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication.

Abbreviations

ATG14/Barkor: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; ER: endoplasmic reticulum; hpi: hours post-infection; IBDV: infectious bursal disease virus; IP: co-immunoprecipitation; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PDPK1: 3-phosphoinositid-dependent protein kinase-1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; vBCL2: viral BCL2 apoptosis regulator.

中文翻译：

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Avibirnavirus VP3 与 PIK3C3-PDPK1 复合物的结合通过激活 AKT-MTOR 通路抑制自噬。

巨自噬/自噬是宿主的自然防御反应。病毒已经开发出各种策略来在其生命周期中破坏自噬。最近，我们发现Avibirnavirus与细胞结合激活了自噬。在本研究中，我们报告了 PIK3C3/VPS34 通过依赖 PDPK1 的 AKT-MTOR 通路对自噬的抑制。自噬检测揭示病毒蛋白VP3在Avibirnavirus早期触发自噬抑制复制。随后的相互作用分析表明，VP3 的 CC1 结构域通过与 BECN1 直接相互作用解离 PIK3C3-BECN1 复合物并阻止自噬体形成，而 VP3 的 CC3 结构域通过直接结合 PIK3C3 破坏 PIK3C3-PDPK1 复合物并抑制自噬体的形成和成熟。此外，我们发现 PDPK1 激活 AKT-MTOR 通路，通过与 AKT 结合来抑制自噬。最后，我们证明了 CC3 域对于 VP3通过自噬调节Avibirnavirus复制的作用至关重要。总之，我们的研究发现Avibirnavirus VP3 将 PIK3C3-PDPK1 复合物与 AKT-MTOR 通路联系起来并抑制自噬，这是控制病毒复制的关键步骤。

缩写

ATG14/Barkor：自噬相关14；BECN1: beclin 1; CC：盘绕线圈；ER：内质网；hpi：感染后的小时数；IBDV：传染性法氏囊病病毒；IP：共免疫沉淀；mAb：单克隆抗体；MAP1LC3/LC3：微管相关蛋白1轻链3；MOI：感染复数；MTOR：雷帕霉素激酶的机制靶点；PDPK1：3-磷酸肌醇依赖性蛋白激酶-1；PIK3C3/VPS34：磷脂酰肌醇3-激酶催化亚基3型；PtdIns3K：磷脂酰肌醇3-激酶；PtdIns3P：3-磷酸磷脂酰肌醇；SQSTM1：螯合体 1；vBCL2：病毒 BCL2 细胞凋亡调节剂。