Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.神经坏死病毒（nervous necrosis virus, NNV）可感染多种重要海淡水经济鱼类，给水产养殖业造成巨大经济损失。自噬是一种进化上保守的依赖于溶酶体的细胞物质降解途径，参与多种病毒感染过程。研究表明，NNV能够诱导鱼类细胞发生自噬促进自身复制，但NNV在入侵阶段诱导细胞自噬的分子机制尚不清楚。在该研究中，我们发现在2种鱼类细胞中赤点石斑鱼神经坏死病毒（red-spotted grouper NNV，RGNNV）都能够在感染1.5和3 h时诱导自噬小体形成，表明RGNNV入侵阶段即可诱发细胞自噬。进一步研究发现RGNNV感染能够抑制自噬溶酶体和自噬小体融合，表明RGNNV入侵阶段诱导细胞发生不完全自噬。随后研究发现RGNNV表面的唯一结构蛋白衣壳蛋白（capsid protein，CP）通过与RGNNV受体海鲈热休克蛋白90ab1(heat shock protein HSP90ab1，LjHSP90ab1)结合抑制AKT-MTOR信号通路，从而诱导细胞自噬发生。机制研究表明CP蛋白竞争性结合于LjHSP90ab1的NM结构域以阻碍AKT与LjHSP90ab1的结合，从而抑制AKT-MTOR信号通路，诱导细胞自噬发生。综上所述，RGNNV在入侵阶段利用CP结合其受体HSP90ab1，进而抑制AKT-MTOR信号通路，诱导细胞不完全自噬。该研究揭示了NNV受体在病毒感染诱导细胞自噬中的功能，为深入解析NNV感染的致病机制提供了新视角。.