Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome.

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.

Characterizing the effects of insertion of a 5.2 kb region of a VACV genome, which contains known immune evasion genes, on MVA immunogenicity.

Modified Vaccinia virus Ankara (MVA) is an attenuated Vaccinia virus (VACV) that is a popular vaccine vector candidate against many different pathogens. Its replication-restricted nature makes it a safe vaccine. However, higher doses or multiple boosts of MVA are necessary to elicit an immune response similar to wild-type VACV. Multiple strategies have been used to create modified MVA viruses that remain safe, but have increased immunogenicity. For example, one common strategy is to delete MVA immunomodulatory proteins in hopes of increasing the host immune response. Here, we take the opposite approach and examine, for the first time, how re-introduction of a 5.2 kb region of VACV DNA (that codes for multiple immunomodulatory proteins) into MVA alters viral immunogenicity. Since antigen presenting cells (APCs) are critical connectors between the innate and adaptive immune system, we examined the effect of MVA/5.2 kb infection in these cells in vitro. MVA/5.2 kb infection decreased virus-induced apoptosis and virus-induced NF-κB activation. MVA.5.2 kb infection decreased TNF production. However, MVA/5.2 kb infection did not alter APC maturation or IL-6 and IL-8 production in vitro. We further explored MVA/5.2 kb immunogenicity in vivo. VACV-specific CD8+ T cells were decreased after in vivo infection with MVA/5.2 kb versus MVA, suggesting that the MVA/5.2 kb construct is less immunogenic than MVA. These results demonstrate the limitations of in vitro studies for predicting the effects of genetic manipulation of MVA on immunogenicity. Although MVA/5.2 kb did not enhance MVA's immunogenicity, this study examined an unexplored strategy for optimizing MVA, and the insight gained from these results can help direct how to modify MVA in the future.

The Molluscum Contagiosum Virus protein MC163 localizes to the mitochondria and dampens mitochondrial mediated apoptotic responses.

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.

Voluntary Wheel Running Does Not Alter Mortality to or Immunogenicity of Vaccinia Virus in Mice: A Pilot Study.

Exercise has been shown to improve immune responses to viral infections and vaccines in several mouse models. However, previous pathogen studies have primarily used infections limited to the respiratory tract. Additionally, previous studies have utilized forced treadmill exercise paradigms, and voluntary wheel running (VWR) has been shown to have differential effects on the immune system in non-infection models. We examined whether VWR could improve morbidity and mortality to a 50% lethal dose of vaccinia virus (VACV), a systemic pathogen commonly used to examine immune responses. Additionally, we examined whether VWR could improve antibody response to a replication-deficient strain of VACV, mimicking a vaccination. Male C57Bl/6J mice underwent 8 weeks of VWR or remained sedentary, then were infected intranasally with 105 PFU VACV strain WR and followed 14 days for weight loss. Mice in the vaccination study ran or were sedentary for 8 weeks, then were given 106 PFU of replication-deficient VACV strain MVA intraperitoneally. Blood was collected at 1, 2, and 4 weeks post-inoculation, and anti-VACV IgG titer was determined by ELISA. VWR did not improve mortality due to VACV infection (p = 0.26), although fewer VWR mice (4/10) died compared to sedentary (SED, 6/10). VWR did not prevent body weight loss due to infection compared to SED (p = 0.20), although VWR mice loss slightly less weight compared to SED through the first 6 days post-infection. Food intake was significantly reduced in SED post-infection compared to VWR (p = 0.05). VWR mice developed a greater IgG antibody response, although this was not significant (p = 0.22). In summary, VWR did not protect against mortality to VACV or prevent infection-induced weight loss, and VWR did not enhance antibody responses. However, there were non-significant trends toward VWR-related improvements in these outcomes, and post-infection food intake was improved by VWR.