Apoptosis is a powerful host cell defense to prevent viruses from completing replication. Poxviruses have evolved complex means to dampen cellular apoptotic responses. The poxvirus, Molluscum Contagiosum Virus (MCV), encodes numerous host interacting molecules predicted to antagonize immune responses. However, the function of the majority of these MCV products has not been characterized. Here, we show that the MCV MC163 protein localized to the mitochondria via an N-terminal mitochondrial localization sequence and transmembrane domain. Transient expression of the MC163 protein prevented mitochondrial membrane permeabilization (MMP), an event central to cellular apoptotic responses, induced by either Tumor Necrosis Factor alpha (TNF-α) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). MC163 expression prevented the release of a mitochondrial intermembrane space reporter protein when cells were challenged with TNF-α. Inhibition of MMP was also observed in cell lines stably expressing MC163. MC163 expression may contribute to the persistence of MCV lesions by dampening cellular apoptotic responses.

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.

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