MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Detection Center of Terrestrial Wildlife Disease, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural Universitygrid.27871.3b, Nanjing, Jiangsu, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affairs, National Center for Engineering Research of Veterinary Bio-Products, Nanjing, China.
J Virol. 2022 Aug 10;96(15):e0080722. doi: 10.1128/jvi.00807-22. Epub 2022 Jul 19.
Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.
禽腺病毒血清型 4（FAdV-4）感染导致肉鸡发生严重的肝炎-心包积水综合征（HHS），给家禽业造成了巨大的经济损失；然而，FAdV-4 感染宿主的确切反应仍不清楚。在本研究中，我们通过液相色谱-串联质谱（LC-MS/MS）检测鉴定了主要病毒蛋白（Hexon、Fiber 1、Fiber 2 和 Penton 基）与宿主蛋白之间的 141 个高可信度的蛋白质-蛋白质相互作用（PPIs）。我们发现，热休克蛋白 70（Hsp70）——得分最高的蛋白及其辅助因子 DnaJ 热休克蛋白 40 家族成员 C7（DnaJC7）——可以负调控 FAdV-4 的复制。此外，Hsp70 的核苷酸结合域（NBD）和 DnaJC7 的 J 结构域对于抑制 FAdV-4 的复制是必需的。我们验证了 DnaJC7 作为桥梁可以与 Hsp70 和 Hexon 结合，协助 Hsp70 和 Hexon 之间的间接相互作用。此外，我们发现 FAdV-4 感染以剂量依赖的方式强烈诱导自噬蛋白和细胞内 Hsp70 的表达。当自噬途径被特异性抑制剂氯喹（CQ）阻断时，通过过表达 Hsp70 阻断 Hexon 的作用显著降低。我们的结果表明，Hsp70 被 DnaJC7 募集来与病毒 Hexon 相互作用，并通过自噬途径抑制 Hexon，从而显著限制了 FAdV-4 的复制。FAdV-4 作为 HHS 的主要原因，近年来在世界范围内迅速传播，严重威胁着家禽业。本研究的目的是鉴定可能调节 FAdV-4 生命周期的重要宿主蛋白。我们发现 Hsp70 和 DnaJC7 在调节病毒 Hexon 的数量和细胞外病毒滴度方面发挥了关键作用。此外，我们证明 Hsp70 在 DnaJC7 的协助下与病毒 Hexon 相互作用，然后通过自噬途径抑制 Hexon 蛋白。这些结果为分子伴侣复合物 Hsp70-DnaJC7 在 FAdV-4 感染中的作用提供了新的见解，并为通过靶向 Hsp70、DnaJC7 和 Hexon 之间的特定相互作用开发抗 FAdV-4 药物提供了新的策略。
