Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity

Macroautophagy/autophagy is an essential cellular response in the fight against intracellular pathogens. Although some viruses can escape from or utilize autophagy to ensure their own replication, the responses of autophagy pathways to viral invasion remain poorly documented. Here, we show that peste des petits ruminants virus (PPRV) infection induces successive autophagic signalling in host cells via distinct and uncoupled molecular pathways. Immediately upon invasion, PPRV induced a first transient wave of autophagy via a mechanism involving the cellular pathogen receptor NECTIN4 and an AKT-MTOR-dependent pathway. Autophagic detection showed that early PPRV infection not only increased the amounts of autophagosomes and LC3-II but also downregulated the phosphorylation of AKT-MTOR. Subsequently, we found that the binding of viral protein H to NECTIN4 ultimately induced a wave of autophagy and inactivated the AKT-MTOR pathway, which is a critical step for the control of infection. Soon after infection, new autophagic signalling was initiated that required viral replication and protein expression. Interestingly, expression of IRGM and HSPA1A was significantly upregulated following PPRV replication. Strikingly, knockdown of IRGM and HSPA1A expression using small interfering RNAs impaired the PPRV-induced second autophagic wave and viral particle production. Moreover, IRGM-interacting PPRV-C and HSPA1A-interacting PPRV-N expression was sufficient to induce autophagy through an IRGM-HSPA1A-dependent pathway. Importantly, syncytia formation could facilitate sustained autophagy and the replication of PPRV. Overall, our work reveals distinct molecular pathways underlying the induction of self-beneficial sustained autophagy by attenuated PPRV, which will contribute to improving the use of vaccines for therapy. <b>Abbreviations:</b> ACTB: actin beta; ANOVA: analysis of variance; ATG: autophagy-related; BECN1: beclin 1; CDV: canine distemper virus; Co-IP: coimmunoprecipitation; FIP: fusion inhibitory peptide; GFP: green fluorescent protein; GST: glutathione S-transferase; HMOX1: heme oxygenase 1; hpi: hours post infection; HSPA1A: heat shock protein family A (Hsp70) member 1A; HSP90AA1: heat shock protein 90 kDa alpha (cytosolic), class A member 1; IFN: interferon; IgG: immunoglobulin G; INS: insulin; IRGM: immunity related GTPase M; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MeV: measles virus; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide-3 kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; SDS: sodium dodecyl sulfate; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UV: ultraviolet.

巨自噬（Macroautophagy）/自噬（autophagy）是宿主对抗胞内病原体的重要细胞应答过程。尽管部分病毒可通过逃逸或劫持自噬完成自身复制，但目前学界对自噬通路应对病毒入侵的调控机制仍知之甚少。本研究发现，小反刍兽疫病毒（PPRV, peste des petits ruminants virus）感染可通过两条独立且互不耦联的分子通路，在宿主细胞中诱导时序性自噬信号激活。感染初期，PPRV通过依赖细胞病原体受体NECTIN4与AKT-MTOR通路的机制，诱导第一波短暂的自噬信号。自噬检测结果显示，PPRV早期感染不仅可提升自噬体与LC3-II的水平，还能下调AKT-MTOR通路的磷酸化水平。后续研究发现，病毒H蛋白与NECTIN4的结合可诱导第二波自噬信号，并使AKT-MTOR通路失活，这一过程是调控病毒感染的关键步骤。感染早期过后，另一类依赖病毒复制与蛋白表达的自噬信号通路被激活。值得注意的是，PPRV复制后，免疫相关GTP酶M（IRGM, immunity related GTPase M）与热休克蛋白家族A（Hsp70）成员1A（HSPA1A, heat shock protein family A (Hsp70) member 1A）的表达水平显著上调。令人惊喜的是，通过小干扰RNA（siRNA, small interfering RNA）敲低IRGM与HSPA1A的表达，会显著削弱PPRV诱导的第二波自噬信号与病毒粒子的产生。此外，分别与IRGM结合的PPRV-C蛋白以及与HSPA1A结合的PPRV-N蛋白的单独表达，即可通过IRGM-HSPA1A依赖通路诱导自噬。重要的是，合胞体的形成可促进持续自噬与PPRV的复制。综上，本研究揭示了减毒PPRV诱导有益持续自噬的具体分子通路，该发现将有助于优化治疗性疫苗的应用。**缩略语：** ACTB：肌动蛋白β（actin beta）；ANOVA：方差分析（analysis of variance）；ATG：自噬相关（autophagy-related）；BECN1：Beclin 1；CDV：犬瘟热病毒（canine distemper virus）；Co-IP：免疫共沉淀（coimmunoprecipitation）；FIP：融合抑制肽（fusion inhibitory peptide）；GFP：绿色荧光蛋白（green fluorescent protein）；GST：谷胱甘肽S-转移酶（glutathione S-transferase）；HMOX1：血红素氧合酶1（heme oxygenase 1）；hpi：感染后小时数（hours post infection）；HSPA1A：热休克蛋白家族A（Hsp70）成员1A（heat shock protein family A (Hsp70) member 1A）；HSP90AA1：热休克蛋白90kDa α（胞质）类A成员1（heat shock protein 90 kDa alpha (cytosolic), class A member 1）；IFN：干扰素（interferon）；IgG：免疫球蛋白G（immunoglobulin G）；INS：胰岛素（insulin）；IRGM：免疫相关GTP酶M（immunity related GTPase M）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；MeV：麻疹病毒（measles virus）；MOI：感染复数（multiplicity of infection）；MTOR：雷帕霉素靶蛋白激酶（mechanistic target of rapamycin kinase）；PI3K：磷酸肌醇3-激酶（phosphoinositide-3 kinase）；PIK3C3：磷脂酰肌醇3-激酶催化亚基3型（phosphatidylinositol 3-kinase catalytic subunit type 3）；SDS：十二烷基硫酸钠（sodium dodecyl sulfate）；siRNA：小干扰RNA（small interfering RNA）；SQSTM1/p62：自噬底物p62（sequestosome 1）；UV：紫外线（ultraviolet）。