WD40结构域是最丰富的结构域之一,并且是真核基因组中的顶级相互作用结构域之一。ATG16L1的WD40结构域对于非规范自噬过程中LC3募集到内溶酶体膜至关重要,但对于规范的自噬是可有可无的。典型的自噬被FMDV利用,而FMDV与非规范自噬之间的关系仍然难以捉摸。在本研究中,通过CRISPR/cas9技术成功产生了WD40敲除(KO)PK15细胞,作为研究非规范自噬对FMDV复制影响的工具。生长曲线分析的结果,形态学观察和核型分析表明,WD40敲除细胞系在生长和形态特征方面是稳定的。感染FMDV后,病毒蛋白的表达,病毒滴度,WD40-KO细胞中病毒RNA的拷贝数显著高于野生型PK15细胞。此外,使用RNA-seq技术对感染或未感染FMDV的WD40-KO细胞和野生型细胞进行测序。差异表达因子Mx1、RSAD2、IFIT1、IRF9、IFITM3、GBP1、CXCL8、CCL5、TNFRSF17显著富集NOD样受体信号通路,RIG-I样受体信号通路,Toll样受体信号通路,细胞因子-细胞因子受体相互作用和TNF信号通路,等。通过qRT-PCR检测差异表达基因的表达水平,这与RNA-seq数据一致。这里,我们首次通过实验证明敲除ATG16L1的WD40结构域可通过下调先天免疫因子来增强FMDV的复制.此外,该结果还表明非规范自噬抑制了FMDV的复制。总的来说,我们的研究结果在调节FMDV的复制水平方面发挥了重要作用,并为病毒-宿主相互作用和潜在的抗病毒策略提供了新的见解.
The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.