Dynamic evolution of MLKL in primate and poxvirus genomes indicates necroptosis is a critical, not auxiliary, countermeasure during infection

Abstract

Pathogen infection triggers host innate defenses which can lead to the activation of regulated cell death (RCD) pathways such as apoptosis. Given a key role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptotic signaling. However, whether this emerging inflammatory RCD pathway functions primarily as a back-up or fundamental response remains inconclusive. We hypothesized that if necroptosis is an instrumental defense, then its effectors should display specific signatures associated with pathogen conflict that are rare in combination: rapid evolution, viral homolog hereafter virolog, and induction by cytokines (e.g. interferons). Our rapid evolution analysis across the necroptosis pathway revealed: 1) strong signatures of positive selection for RIPK3 and MLKL in primate genomes and to a lesser extent DAI/ZBP1, 2) elevated rates of amino acid substitution on multiple surfaces including the RIPK3/MLKL binding interface and 3) evidence supporting a means of activating RIPK3 independent of homotypic RHIM domain interactions. Interestingly, a poxvirus MLKL homolog has recently been identified that acts as a RIPK3 pseudosubstrate. Our findings indicate that poxvirus MLKLs are also subject to similar but distinct volatile patterns of evolution comparable to host necroptotic factors. Specifically, viral MLKLs have undergone numerous gains and losses in poxvirus evolution with some species harboring three distinct copies. Furthermore, we confirm that MLKL can be induced by cytokines like interferon gamma. In summary, MLKL displays all three hallmarks of pivotal immune factors of which only OAS1, but not other factors like cGAS, APOBEC3G, or PKR, exhibits. These data support the hypothesis that over evolutionary time, necroptosis has served as a key battleground during infection and is therefore, not an auxiliary response.

Mono a Mano: ZBP1’s Love–Hate Relationship with the Kissing Virus

“…There is also evidence of host adaptations that could reflect a role for ZBP1 in EBV and other persistent viral infections. In particular, ZBP1 is under positive selection in primates, although the rate is less than that for RIPK3 and MLKL [ 39 , 78 ]. Further evidence of selection is provided by the existence of over 2000 possible ZBP1 splicing isoforms.…”