Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L 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 M1L 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 Saccharomyces cerevisiae 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.

IMPORTANCE 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.

凋亡是重要的抗病毒宿主防御机制。在这里，我们报告鉴定由牛痘病毒（VACV）M1L基因编码的新型凋亡抑制剂。VACV的减毒改良牛痘病毒安卡拉（MVA）株不存在M1L，该株可刺激多种类型免疫细胞的凋亡。M1表达增加了MVA感染的THP-1和Jurkat细胞的活力，并减少了一些凋亡的生化标志，例如PARP-1和procaspase-3的裂解。此外，异位M1L表达降低了星形孢菌素诱导的HeLa细胞内源性凋亡。然后，我们确定了M1抑制功能的分子基础。M1允许线粒体去极化，但阻止procaspase-9加工，表明M1靶向凋亡小体。为了支持该模型，我们发现M1促进了酿酒酵母的存活过度表达人Apaf-1和procaspase-9（凋亡小体的关键组成部分），或仅过表达仅构象活性的caspase-9。在哺乳动物细胞中，M1与Apaf-1–procaspase-9复合物共免疫沉淀。当前的模型是M1与凋亡小体相关并允许其形成，但阻止了凋亡小体的凋亡功能。M1蛋白具有14个预测的锚蛋白（ANK）重复结构域，而M1是报道的第一个使用此抑制策略的含ANK的蛋白。由于含有ANK的蛋白质是由许多大型DNA病毒编码的，并且存在于生活的所有领域，因此对M1的研究可能会导致人们更好地了解ANK蛋白质在病毒与宿主之间的相互作用中的作用。

重要性凋亡选择性地消除了诸如病毒感染细胞之类的危险细胞。痘病毒表达凋亡拮抗剂以中和这种抗病毒宿主防御。牛痘病毒（VACV）M1锚蛋白（ANK）蛋白是一种以前不具有功能的蛋白，可抑制细胞凋亡。M1与凋亡小体相互作用，并在多种细胞类型和多种条件下阻止procaspase-9加工以及下游procaspase-3裂解。M1是第一个与凋亡小体相关并阻止其功能的痘病毒蛋白，可更详细地描述VACV在感染过程中遇到的威胁。细胞凋亡的失调与几种人类疾病有关。凋亡相关疾病的一种潜在治疗方法是使用类似于M1的设计的ANK重复蛋白（DARPins）作为半胱天冬酶抑制剂。