PROJECT SUMMARY Poxviruses include family members that are categorized as Class A Priority Pathogens with significant future pandemic potential, while modified forms of other family members are widely used as vaccine vectors and oncolytic therapeutics. Beyond their more direct medical significance, poxviruses also have a long history as invaluable research tools at the forefront in the discovery of fundamental cellular processes. These include the discovery of 5’ 7-Methyl GTP capping, 2’-O-Methylation and 3’ polyadenylation that we now know to occur on almost all eukaryotic as well as viral mRNAs. This is in part because poxviruses encode their own fully functional DNA replication, transcription and mRNA biogenesis machinery, which enables these remarkably self-sufficient DNA viruses to replicate in the cytoplasm. Despite this, like all other viruses, poxviruses remain entirely dependent on gaining access to host ribosomes to synthesize viral proteins and replicate. While the mechanisms by which both viruses and their host cells control translation through the activity of eukaryotic initiation factors (eIFs) are well understood, until recently ribosomes were largely viewed as passive code- reading machines that lacked intrinsic regulatory capacity. In the prior award, we revealed that the poxvirus kinase, B1 phosphorylates a number of ribosomal subunit proteins (RPs) at Serine and Threonine residues that are not modified in uninfected cells or cells infected with other viruses. These include phosphorylation of S278 in a loop domain of the small 40S RP, Receptor for Activated C Kinase 1 (RACK1) that serves to enhance translation of viral mRNAs that harbor unusual 5’ polyA-leaders. Such leaders are normally selected against in their mammalian hosts where adenosine homopolymers are restricted to the 3’ untranslated polyA- tail, but B1-mediated phosphorylation effectively mimics negatively charged amino acids that are found in the RACK1 loop of dicot plants and protists whose translation systems naturally accommodate 5’ polyA. Beyond insights into viral manipulation of ribosomes, these findings provided one of the first examples of ribosomal post-translational modifications that can regulate translation directly as well as unexpected insights into the structural and functional diversification of ribosomes across different species. Beyond RACK1, which lies at the mRNA exit channel, we also identified additional poxvirus-specific phosphorylation events in other small RPs, including RPS28 that lies at the mRNA entry channel. Biochemical and structure modeling lead us to hypothesize that these modified RPs form an interconnected network whose phosphorylation enables poxviruses to remodel the mRNA channel of the ribosome to better accommodate 5’ polyA-leaders in their mammalian hosts. Preliminary cryo electron microscopy (cryo-EM) and functional studies using a newly developed cell system reconstituted with phosphomimetics of poxvirus-modified RPs further...