Macroautophagy/autophagy plays an essential role in cellular responses to pathogens. However, the precise mechanisms and signaling pathways that modulate cellular autophagy in classical swine fever virus (CSFV)-infected host cells have not been confirmed. In this study, we showed that CSFV infection inhibits the phosphorylation of MTOR (mechanistic target of rapamycin kinase), subsequently leading to autophagy initiation. We also show that MAPK/ERK (mitogen-activated protein kinase) signaling is involved in CSFV-induced autophagy. The CSFV-induced inhibition of AKT/PKB (AKT serine/threonine kinase)-MTOR was observed to be partially responsible for the MTOR inactivation and subsequent autophagy initiation. Moreover, the CAMKK2/CaMKKβ (calcium/calmodulin dependent protein kinase kinase 2)-PRKAA/AMPK (protein kinase AMP-activated catalytic subunit alpha) axis was found to be involved in CSFV-induced autophagy. Meanwhile, CSFV non-structural protein NS5A induced autophagy via the CAMKK2-PRKAA-MTOR signaling pathway but not the AKT-MTOR or MAPK1/ERK2-MAPK3/ERK1-MTOR pathway. Although the AKT-MTOR pathway also plays an important role in the induction of autophagy by CSFV. We also found the interaction between HSP90AB1/HSPCB and NS5A by tandem affinity purification/liquid chromatography-mass spectrometry (LC-MS) and immunoprecipitation. Furthermore, the CSFV-induced [Ca²⁺]_cyto increase potently induced autophagy through CAMKK2 and PRKAA. Moreover, we isolated and identified the BECN1/Beclin 1 protein complexes by tandem affinity purification/LC-MS and immunoprecipitation, the interaction between BECN1 and MAVS was confirmed by immunoprecipitation, laser scanning confocal microscope technology, and GST affinity-isolation experiments. Furthermore, CSFV-mediated autophagy suppressing type I IFN production is related to the interaction between MAVS and BECN1. Finally, the modulation of autophagy induction pathways by different autophagy regulatory factors significantly affected the replication of CSFV.

Abbreviations: AKT: AKT serine/threonine kinase; AMPK: Adenosine monophosphate-activated protein kinase; CAMKK2: Calcium/calmodulin dependent protein kinase kinase 2; CSFV: Classical swine fever virus; HRP: Horseradish peroxidase; HSP90AB1: Heat shock protein 90 alpha family class B member 1; IFN: Interferon; ISGs: IFN-stimulated genes; LC-MS: Liquid chromatography-mass spectrometry; MAP1LC3/LC3: Microtubule associated protein 1 light chain 3; MAPK: Mitogen-activated protein kinase; MAVS: Mitochondrial antiviral signaling protein; MOI: Multiplicity of infection; MTOR: Mechanistic target of rapamycin kinase; PBS: Phosphate-buffered saline; PRKAA: Protein kinase AMP-activated catalytic subunit alpha; shRNA: short hairpin RNA.

巨自噬/自噬在细胞对病原体的反应中起重要作用。然而，在经典猪瘟病毒 (CSFV) 感染的宿主细胞中调节细胞自噬的精确机制和信号通路尚未得到证实。在这项研究中，我们发现 CSFV 感染抑制 MTOR（雷帕霉素激酶的机制靶标）的磷酸化，随后导致自噬启动。我们还表明 MAPK/ERK（丝裂原活化蛋白激酶）信号参与 CSFV 诱导的自噬。观察到 CSFV 诱导的 AKT/PKB（AKT 丝氨酸/苏氨酸激酶）-MTOR 抑制是 MTOR 失活和随后自噬启动的部分原因。而且，发现 CAMKK2/CaMKKβ（钙/钙调蛋白依赖性蛋白激酶激酶 2）-PRKAA/AMPK（蛋白激酶 AMP 激活的催化亚基 α）轴参与 CSFV 诱导的自噬。同时，CSFV 非结构蛋白 NS5A 通过 CAMKK2-PRKAA-MTOR 信号通路诱导自噬，但不通过 AKT-MTOR 或 MAPK1/ERK2-MAPK3/ERK1-MTOR 通路诱导自噬。尽管 AKT-MTOR 通路在 CSFV 诱导自噬中也发挥着重要作用。我们还通过串联亲和纯化/液相色谱-质谱 (LC-MS) 和免疫沉淀法发现了 HSP90AB1/HSPCB 和 NS5A 之间的相互作用。此外，CSFV 诱导的 [Ca CSFV 非结构蛋白 NS5A 通过 CAMKK2-PRKAA-MTOR 信号通路诱导自噬，但不通过 AKT-MTOR 或 MAPK1/ERK2-MAPK3/ERK1-MTOR 通路诱导自噬。尽管 AKT-MTOR 通路在 CSFV 诱导自噬中也发挥着重要作用。我们还通过串联亲和纯化/液相色谱-质谱 (LC-MS) 和免疫沉淀法发现了 HSP90AB1/HSPCB 和 NS5A 之间的相互作用。此外，CSFV 诱导的 [Ca CSFV 非结构蛋白 NS5A 通过 CAMKK2-PRKAA-MTOR 信号通路诱导自噬，但不通过 AKT-MTOR 或 MAPK1/ERK2-MAPK3/ERK1-MTOR 通路诱导自噬。尽管 AKT-MTOR 通路在 CSFV 诱导自噬中也发挥着重要作用。我们还通过串联亲和纯化/液相色谱-质谱 (LC-MS) 和免疫沉淀法发现了 HSP90AB1/HSPCB 和 NS5A 之间的相互作用。此外，CSFV 诱导的 [Ca²⁺ ]_细胞增加通过 CAMKK2 和 PRKAA 有效诱导自噬。此外，我们通过串联亲和纯化/LC-MS 和免疫沉淀分离和鉴定了 BECN1/Beclin 1 蛋白复合物，免疫沉淀、激光扫描共聚焦显微镜技术和 GST 亲和分离实验证实了 BECN1 和 MAVS 之间的相互作用。此外，CSFV 介导的自噬抑制 I 型 IFN 产生与 MAVS 和 BECN1 之间的相互作用有关。最后，不同自噬调节因子对自噬诱导途径的调节显着影响了CSFV的复制。

缩写： AKT：AKT丝氨酸/苏氨酸激酶；AMPK：腺苷单磷酸活化蛋白激酶；CAMKK2：钙/钙调蛋白依赖性蛋白激酶激酶 2；CSFV：猪瘟病毒；HRP：辣根过氧化物酶；HSP90AB1：热休克蛋白 90 α 家族 B 类成员 1；干扰素：干扰素；ISGs：干扰素刺激基因；LC-MS：液相色谱-质谱法；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MAPK：丝裂原活化蛋白激酶；MAVS：线粒体抗病毒信号蛋白；MOI：感染多样性；MTOR：雷帕霉素激酶的机制靶点；PBS：磷酸盐缓冲盐水；PRKAA：蛋白激酶 AMP 激活的催化亚基 α；shRNA：短发夹RNA。