Divergence in poxvirus-encoded E3-like proteins can dictate poxvirus activation of cellular necroptosis

Poxviruses encode a plethora of proteins adapted to diverse cellular responses against viruses. The poxvirus-encoded E3-like proteins are multifunctional, regulating diverse cellular antiviral responses. The canonical Vaccinia E3-like proteins have two domains: an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD). Using protein sequence and structural homology modeling, we identified dsRNA-BD-fold-containing proteins in all the poxviruses except avipoxviruses, salmon poxvirus, and entomopoxviruses. Furthermore, we show that the acquisition of these proteins likely happened under three distinct events and can be classified into three categories: (i) the E3-like proteins with dsRNA-BD with the presence or absence of the N-terminal domain; (ii) unconventional/putative dsRNA-BD-fold-like proteins, present in macropoxvirus and molluscipoxvirus, and (iii) the dsRNA-BD-containing protein present in crocodile poxvirus. Members of leporipoxvirus, waddenpoxvirus, cetaceanpoxvirus, and selected members of orthopoxvirus contain E3-like proteins missing the N-terminal Zα-BD required for necroptosis inhibition. Using AlphaFold, we also show that the Zα-BD of chordopoxviruses, E3-like proteins, is structurally more variable than the dsRNA-binding domain. Compared to orthopoxviruses that inhibit necroptosis and contain an N-terminus Zα-BD, our results show that leporipoxviruses induce receptor-interacting protein kinase (RIPK) RIPK1- and RIPK3-mediated necroptosis in human and mouse necroptosis-competent cell lines. These data suggest that leporipoxviruses lack countermeasures against necroptosis, unlike orthopoxviruses, which encode multiple key regulators of necroptosis, possibly due to a lack of selective pressure in their lagomorph host species.IMPORTANCEThe evolutionary arms race between viruses and their hosts has led to the viral acquisition of genes that antagonize the host's antiviral defenses. However, related viral and host proteins have evolved under positive, purifying, and neutral selection. Poxvirus-encoded E3-like proteins play a pivotal role in regulating the host's cellular antiviral and apoptotic responses. The two domains of canonical E3-like proteins, an N-terminal Z-form nucleic acid-binding domain (Zα-BD) and a C-terminal double-stranded RNA-binding domain (dsRNA-BD), have evolved under varying host immune selection pressures. Here, we demonstrate that the dsRNA-BD is structurally conserved among E3 orthologs, whereas the Zα-BD, involved in regulating necroptosis, exhibits structural diversity and is absent in some poxviruses. Leporipoxviruses, which lack Zα-BD-containing proteins and evolved in species without functional necroptosis, do activate necroptosis in necroptotic-competent cells. Our study thus highlights that a lack of selective pressure from the host can shape viral countermeasures and viral divergence.