Document text
Principal Investigator: Peter Shen
Organization: NORTHWESTERN UNIVERSITY AT CHICAGO
Fiscal Year: 2022
Award: $482,787
Funding agency: National Institute of Allergy and Infectious Diseases
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 supports this
hypothesis and suggests an unprecedented level of ribosome remodeling or “species repurposing” arises from
these phosphorylation events, which this renewal application proposes to study in fine mechanistic detail.
Terms: <Adenosine><Amino Acids><Award><Binding><Biochemical><Biogenesis><Biology><Category A pathogen><Category A priority pathogen><Cell Body><Cell Function><Cell Process><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular biology><Charge><Code><Coding System><Cryo-electron Microscopy><Cryoelectron Microscopy><Custom><Cytoplasm><Cytosol><DNA Replication><DNA Synthesis><DNA Viruses><DNA biosynthesis><Double Stranded DNA Virus><Electron Cryomicroscopy><Eukaryotic Initiation Factors><Eukaryotic Peptide Initiation Factors><Eukaryotic Translation Initiation Factors><Event><Evolution><Face><Family member><Future><GTP><Gene Transcription><Genetic Transcription><Genetic Translation><Goals><Guanosine Triphosphate><History><Human><In Vitro><Kinases><L-Serine><L-Threonine><Laboratories><Lead><Left><Mediating><Medical><Messenger RNA><Methylation><Modeling><Modern Man><Molecular Interaction><Oncolytic><Oranges><Origin of Life><P variolae><P. variolae><P.variolae><Pb element><Phosphates><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Phylogenetic Analysis><Phylogenetics><Plants><Polyadenylation><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Poxviridae><Poxvirus officinale><Poxvirus variolae><Poxviruses><Protein Biosynthesis><Protein Modification><Protein Phosphorylation><Protein Subunits><Proteins><RNA Expression><RNA Polyadenylation><RPS3><RPS3 gene><Reading><Recording of previous events><Research><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Ribosomes><Serine><Signaling Molecule><Site><Smallpox><Smallpox Vaccine><Smallpox virus><Structure><Subcellular Process><Surface><System><Tail><Testing><Therapeutic><Threonine><Transcription><Translations><Transphosphorylases><Vaccination><Vaccinia virus><Variola><Variola major virus><Variola virus><Viral><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virus><Virus Replication><Zoonoses><Zoonotic><Zoonotic Infection><aminoacid><cell biology><cryo-EM><cryoEM><dsDNA Virus><faces><facial><heavy metal Pb><heavy metal lead><inorganic phosphate><insight><mRNA><mRNA Translation><mRNA capping><pandemic><pandemic disease><plasmid vaccine><pox virus><priority pathogen><programs><protein function><protein kinase C receptor><protein synthesis><prototype><receptors for activated C kinase><recombinant vaccinia virus><reconstitute><reconstitution><small pox><small pox vaccine><small pox virus><tool><variola major><vector vaccine><viral multiplication><viral replication><virus multiplication><virus protein>