UC San Diego

Despite being a critical component of the antiviral response, inflammatory signaling pathways are known to be hijacked by pathogens from diverse viral families, resulting in poor health outcomes for infected hosts. Receptor interacting protein kinase 3 (RIPK3) is a key inflammatory mediator traditionally studied for its role facilitating apoptosis and necroptosis, a highly inflammatory and lytic cell death pathway. During viral infection, RIPK3 dysregulation contributes to increased virus replication. In fact, enterovirus-encoded 3C proteases are known to cleave RIPK3 to disrupt activation of necroptosis. However, in addition to mediating cell death, RIPK3 is also associated with cell survival and activation of the nuclear factor kappa B (NF-κB) transcription factor; Upon phospho-activation in the cytoplasm, the NF-κB complex translocates to the nucleus, stimulating the production of pro-inflammatory cytokines. Whilst viruses are known to dysregulate inflammatory cell death signaling from RIPK3, the implications of cleavage on NF-κB activation are extremely understudied. Moreover, RIPK3 is known to be undergoing rapid evolution in primates, with over 20 residues showing significant rates of mutation throughout the protein, including some in close proximity to the enterovirus cut site. Using an in vitro bioluminescent reporter assay, we show that the enterovirus 3C protease will reduce NF-κB activation through cleavage of RIPK3 when overexpressed in HEK293T cells. Moreover, we show that primate amino acid residues at and around the enterovirus cut site confer protection from viral cleavage of RIPK3 and restores NF-κB activity. Finally, we demonstrate that viral antagonism of RIPK3 orthologues from a diverse array of vertebrate species is well conserved. The results of our study demonstrate the importance of inhibiting RIPK3-induced inflammatory signaling for enterovirus pathogenesis, and the protective diversity in RIPK3 sequence seen in primates suggests a host-virus arms race interaction between RIPK3 and enteroviruses, explaining the rapid evolution observed with this protein.