RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells.
RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells.
复制标题
RIPK3 依赖性坏死性凋亡限制 PK-15 细胞中的 PRV 复制
DOI：
10.3389/fmicb.2021.664353
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发表时间：
2021
影响因子：
5.2
通讯作者：
Li C
中科院分区：
文献类型：
--
作者：
Gou H;Bian Z;Cai R;Chu P;Song S;Li Y;Jiang Z;Zhang K;Yang D;Li C
感染伪狂犬病病毒(PRV)的猪表现为多器官坏死性病变。PRV诱导细胞死亡的机制尚不清楚。近年来，坏死性上睑下垂被认为是一种依赖于受体相互作用蛋白激酶3(RIPK3)和混合谱系蛋白样蛋白(MLKL)的程序性过程。在这项研究中，我们证明了PRV在PK-15细胞中诱导了RIPK3依赖的坏死性下垂。碘化丙啶(PI)阳性染色显示PRV感染引起细胞死亡。透射电子显微镜分析表明，PRV感染的细胞质膜破裂。泛半胱氨酸氨基转移酶抑制剂不能阻止PRV诱导的坏死性细胞死亡。Western印迹分析表明，在PRV感染过程中，caspase-3和caspase-8没有被切割。虽然PRV感染后肿瘤坏死因子-α的转录增加，但其特异性抑制物不参与PRV诱导的坏死性细胞死亡。进一步的实验表明，RIPK3和MLKL的磷酸化在PRV感染的细胞中上调。稳定的shRNA敲除RIPK3或MLKL对PRV诱导的坏死性细胞死亡有恢复作用。同时，病毒滴度在RIPK3和MLKL基因敲除细胞中升高。因此，我们得出结论，在宿主细胞中启动坏死性下垂在PRV感染中起着有限的作用。考虑到坏死性下垂是一种炎性形式的程序性细胞死亡，我们的数据可能有助于理解感染PRV的猪的坏死性病理。
Pigs infected by pseudorabies virus (PRV) display necrotic pathology in multiple organs. The mechanism by which PRV induces cell death is still unclear. Recently, necroptosis was identified as a programmed process dependent on the receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase-like protein (MLKL). In this study, we demonstrated that PRV induced RIPK3-dependent necroptosis in PK-15 cells. The data showed that PRV infection caused cell death with Propidium Iodide (PI)-positive staining. Transmission electron microscopy analysis indicated plasma membrane disruption in PRV-infected cells. A pan-caspase inhibitor did not prevent PRV-induced necrotic cell death. Western blot analysis indicated that caspase-3 and caspase-8 were not cleaved during PRV infection. Although the transcription of tumor necrosis factor-alpha (TNF-α) was increased by PRV infection, RIPK1 was shown to be not involved in PRV-induced necrotic cell death by use of its specific inhibitor. Further experiments indicated that the phosphorylation of RIPK3 and MLKL was upregulated in PRV-infected cells. Stable shRNA knockdown of RIPK3 or MLKL had a recovery effect on PRV-induced necrotic cell death. Meanwhile, viral titers were enhanced in RIPK3 and MLKL knockdown cells. Hence, we concluded that initiation of necroptosis in host cells plays a limiting role in PRV infection. Considering that necroptosis is an inflammatory form of programmed cell death, our data may be beneficial for understanding the necrotic pathology of pigs infected by PRV.
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