Ryerson Melissa R, Richards Monique M, Kvansakul Marc, Hawkins Christine J, Shisler Joanna L
Department of Microbiology, University of Illinois, Urbana, Illinois, USA.
Department of Biochemistry and Genetics, La Trobe University, Melbourne, Australia.
J Virol. 2017 Nov 14;91(23). doi: 10.1128/JVI.01385-17. Print 2017 Dec 1.
Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) gene. is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions. Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.
细胞凋亡是一种重要的抗病毒宿主防御机制。在此，我们报告鉴定出一种由痘苗病毒（VACV）基因编码的新型细胞凋亡抑制剂。在VACV的减毒安卡拉痘苗病毒（MVA）株中不存在该抑制剂，MVA株可在多种类型免疫细胞中刺激细胞凋亡。M1的表达增加了受MVA感染的THP - 1细胞和Jurkat细胞的活力，并减少了细胞凋亡的若干生化特征，如PARP - 1和procaspase - 3的切割。此外，异位表达降低了HeLa细胞中星形孢菌素诱导的（内源性）细胞凋亡。然后，我们确定了M1抑制功能的分子基础。M1可使线粒体去极化，但阻断procaspase - 9加工，这表明M1靶向凋亡小体。支持该模型的是我们发现，在过表达人Apaf - 1和procaspase - 9（凋亡小体的关键组分）或仅过表达构象活性caspase - 9的细胞中，M1可促进细胞存活。在哺乳动物细胞中，M1与Apaf - 1 - procaspase - 9复合物共免疫沉淀。目前的模型是，M1与凋亡小体结合并允许其形成，但阻止凋亡小体的凋亡功能发挥。M1蛋白具有14个预测的锚蛋白（ANK）重复结构域，且M1是首个被报道采用这种抑制策略的含ANK蛋白。由于含ANK蛋白由许多大型DNA病毒编码且存在于生命的所有域中，对M1的研究可能有助于更好地理解ANK蛋白在病毒 - 宿主相互作用中的作用。细胞凋亡可选择性地清除危险细胞，如病毒感染细胞。痘病毒表达细胞凋亡拮抗剂以中和这种抗病毒宿主防御。痘苗病毒（VACV）的M1锚蛋白（ANK）蛋白是一种先前未赋予功能的蛋白，可抑制细胞凋亡。M1在多种细胞类型和多种条件下与凋亡小体相互作用，并阻止procaspase - 9加工以及下游procaspase - 3切割。M1是首个被报道与凋亡小体结合并阻止其功能发挥的痘病毒蛋白，这更详细地描绘了VACV在感染过程中所面临的威胁。细胞凋亡失调与多种人类疾病相关。一种潜在的治疗凋亡相关疾病的方法是使用类似于M1的设计锚蛋白重复蛋白（DARPins）作为caspase抑制剂。因此，通过凋亡小体结合来研究M1的新型抗凋亡作用将有助于理解如何利用天然或合成分子来控制细胞凋亡。
