Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3

Abstract

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3

“…The importance of necroptosis in host defense is supported by the observation that the genomes of poxviruses, herpesviruses, and cytomegaloviruses encode proteins that target the necroptosis machinery, thereby conferring a survival advantage by blocking an altruistic cell death mechanism. Most viral effectors reported to block necroptosis target the RHIM sequences of RIPK1, RIPK3, and the RHIM domain-containing viral RNA sensor ZBP1/DNA-dependent activator of interferon regulatory factors (DAIs) (Guo et al, 2015a(Guo et al, , 2015bKoehler et al, 2017;Upton et al, 2010Upton et al, , 2012. By targeting the RHIM, a $16-residue oligomerization module, pathogens can block necrosome assembly and halt downstream necroptosis signaling.…”

The involvement of regulated cell death forms in modulating the bacterial and viral pathogenesis

“…Along with this, E3 deleted VACV strain triggers immediate necroptosis in interferon treated mouse L292 and human embryonal kidney (HEK293T) cells. The cytotoxic effect of the engineered VACV strain is attributed to necroptotic signaling, because the pathogenicity of the virus can be restored in vivo by the depletion of either RIPK3 or ZBP1 in mice (Koehler et al, 2017).…”

The search for a PKR code—differential regulation of protein kinase R activity by diverse RNA and protein regulators

“…E3L. Poxviruses rely on the multifunctional Vaccinia virus E3L protein to escape host innate immune response and to achieve full pathogenicity (Langland and Jacobs 2002;Kwon and Rich 2005;Koehler et al 2017). E3L contains an amino-terminal Z-DNA binding domain (ZBD) followed by a carboxy-terminal dsRBM (Ha et al 2004).…”