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- Genre: Doctoral Dissertation
Description
Monkeypox virus (MPXV) is an orthopoxvirus that causes smallpox-like disease and has up to a 10% mortality rate, depending on the infectious strain. The global eradication of the smallpox virus has led to the decrease in smallpox vaccinations, which has led to a drastic increase in the number of human MPXV cases. MPXV has been named the most important orthopoxvirus to infect humans since the eradication of smallpox and has been the causative agent of the 2022 world-wide MPXV outbreak. Despite being highly pathogenic, MPXV contains a natural truncation at the N-terminus of its E3 homologue. Vaccinia virus (VACV) E3 protein has two domains: an N- terminus Z-form nucleic acid binding domain (Z-BD) and a C-terminus double stranded RNA binding domain (dsRBD). Both domains are required for pathogenesis, interferon (IFN) resistance, and protein kinase R (PKR) inhibition. The N-terminus is required for evasion of Z-DNA binding protein 1 (ZBP1)-dependent necroptosis. ZBP1 binding to Z- form deoxyribonucleic acid/ribonucleic acid (Z-DNA/RNA) leads to activation of receptor-interacting protein kinase 3 (RIPK3) leading to mixed lineage kinase domain- like (MLKL) phosphorylation, aggregation and cell death. This study investigated how different cell lines combat MPXV infection and how MPXV has evolved ways to circumvent the host response. MPXV is shown to inhibit necroptosis in L929 cells by degrading RIPK3 through the viral inducer of RIPK3 degradation (vIRD) and by inhibiting MLKL aggregation. Additionally, the data shows that IFN treatment efficiently inhibits MPXV replication in a ZBP1-, RIPK3-, and MLKL- dependent manner, but independent of necroptosis. Also, the data suggests that an IFN inducer with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections. Furthermore, it reveals a link between PKR and pathogen-induced necroptosis that has not been previously described.
ContributorsWilliams, Jacqueline (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2022
Description
Environmental stressors can perturb cellular homeostasis. Cells activate an integrated stress response that will alleviate the effects of the ongoing stress. Stress-activated protein kinases function to phosphorylate the eukaryotic translation initiation factor, eIF2α, which results in inhibition of translation of house-keeping genes. Following these events, formation of cytoplasmic messenger ribonucleoprotein complexes, known as stress granules, will take place. Stress granules typically have a pro-survival function. These studies demonstrate that assembly of stress granules can also lead to necroptosis. Necroptosis is a caspase-independent, receptor-interacting protein kinase 3 (RIPK3)-dependent cell death pathway executed by mixed lineage kinase domain-like (MLKL) protein. Cellular stress is induced using arsenite (oxidative stress) or by infection with vaccinia virus (VACV) E3 protein Z-DNA-binding domain mutant, VACV-E3LΔ83N. In both cases, RIPK3-dependent death was observed in interferon (IFN)-primed L929 cells. This death led to phosphorylation and trimerization of MLKL, indicative of necroptosis. Necroptosis induced by oxidative stress and VACV-E3LΔ83N infection was dependent on the host Z-form nucleic acid sensor, DNA-dependent activator of IFN-regulatory factors (DAI), as it was inhibited in DAI-deficient L929 cells. Under both cellular stresses, DAI associated with RIPK3 and formed high-molecular-weight complexes, consistent with formation of the necrosomes. DAI localized into stress granules during necroptosis induced by arsenite and the mutant virus, and the necrosomes formed only in presence of stress granule assembly. The significance of stress granules for cellular stress-induced necroptosis was demonstrated using knock-out (KO) cell lines unable to form granules: T cell-restricted intracellular antigen 1 (TIA-1) KO MEF cells and Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1/2) KO U2OS cells. Necroptosis was inhibited in absence of stress granule formation as no cell death or activation of MLKL was observed in the knock-out cell lines following arsenite treatment or VACV-E3LΔ83N infection. Furthermore, wild-type VACV was able to inhibit stress granule assembly, which coincided with the virus ability to inhibit necroptosis. These studies have led to a model of Z-form nucleic acids being involved in activation of the stress granule-mediated necroptosis following induction by environmental stressors. These results have significance for understanding the etiology of human diseases and the antiviral innate immunity.
ContributorsSzczerba, Mateusz Bartlomiej (Author) / Jacobs, Bertram L (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2021