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Principal Investigator: Peter Shen
Organization: NORTHWESTERN UNIVERSITY AT CHICAGO
Fiscal Year: 2024
Award: $678,119
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT
Although smallpox was formally declared to have been eradicated in 1980, molluscum contagiosum is
widespread and a variety of zoonotic poxviruses continually infect and adapt to humans. This is exemplified by
monkeypox/Mpox virus, which causes outbreaks in humans with increasing frequency and resulted in a global
outbreak and declaration of a new WHO poxvirus emergency in 2022. Yet several other poxviridae family
members are used as vaccine vectors and oncolytics. Beyond their direct medical significance, studies of
poxviruses have a long history of providing new insights into fundamental aspects of cell biology and
immunology, due in part to their unusual replication cycle and complex immune evasion strategies. Other than
the singular, related African Swine Fever Virus, poxviruses are the only mammalian DNA viruses that replicate
entirely in the cytoplasm. To do this, poxviruses encode their own fully functional DNA replication, transcription
and mRNA biogenesis machinery, forming large cytoplasmic replication sites called “viral factories”. Despite
this, poxviruses remain dependent upon their host cell’s mRNA translation machinery and metabolic pathways
to complete their replication cycle, while their mode of replication makes them highly vulnerable to cytosolic
sensors aimed at detecting their presence and mounting antiviral responses. These metabolic and sensing
processes are intertwined yet how poxviruses control them is both complex and poorly understood. Through
co-immunoprecipitation and mass spectrometry-based screening in biologically relevant primary cells, we
discovered that a highly conserved poxvirus protein, called F17, targets the central metabolic sensor and
effector kinase, mammalian/mechanistic Target of Rapamycyin (mTOR) in unique ways. Unlike other viruses
that target upstream signaling to mTOR to indirectly stimulate or repress its activity, we find that F17 directly
targets the two distinct mTOR Complexes 1 and 2 (mTORC1, mTORC2) to “dysregulate” their activity. This is
achieved through F17 binding to unique N-terminal conserved domains in the mTOR regulatory subunits,
Raptor and Rictor, resulting in their competitive sequestration from binding to mTOR. Moreover, we find that
F17 is required to block Interferon Stimulated Gene (ISG) responses that are initiated by the cytosolic sensor,
cGAS. While the precise nature of these host responses and how F17 counteracts them remains unclear,
additional preliminary data suggests that while other viral proteins function to counteract cGAS-mediated
responses to viral DNA, F17 instead blocks cGAS-mediated responses that are driven by mitochondrial DNA
release, and which require mTOR-mediated metabolic rewiring to drive ISG production. This proposal will
determine the structural basis of mTOR dysregulation by F17, how this contributes to virus replication and
spread in various biologically relevant human cell types, and how F17 counteracts mitochondrial-driven
antiviral responses. Upon completion, this proposal will illuminate previously unrecognized aspects of innate
responses and viral countermeasures that occur during poxvirus infection.
Terms: <African Swine Fever Virus><Agonist><Anti-viral Response><Antigen-Antibody Complex><Binding><Biogenesis><Category A pathogen><Category A priority pathogen><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Cellular biology><Citric Acid Cycle><Co-Immunoprecipitations><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><DNA><DNA Replication><DNA Synthesis><DNA Viruses><DNA biosynthesis><Data><Deoxyribonucleic Acid><Detection><Disease Outbreaks><Electron Cryomicroscopy><Emergencies><Emergency Situation><Equilibrium><FK506 Binding Protein 12-Rapamycin Associated Protein 1><FKBP12 Rapamycin Complex Associated Protein 1><FRAP1><FRAP1 gene><FRAP2><Family><Family member><Frequencies><Gene Expression><Gene Transcription><Genes><Genetic Transcription><Glycolysis><History><Human><IFN><Immune><Immune Complex><Immune Evasion><Immune response><Immunes><Immunological response><Immunology><Infection><Interferons><Intracellular Communication and Signaling><Kinases><Krebs Cycle><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mechanistic Target of Rapamycin><Mediating><Medical><Messenger RNA><Metabolic><Metabolic Pathway><Mitochondria><Mitochondrial DNA><Modeling><Modern Man><Molecular Interaction><Molluscum Contagiosum><Monkey Pox><Monkey Pox Virus><Monkeypox><Monkeypox virus><Monkeypoxvirus><N-terminal><NH2-terminal><Nature><Oncolytic><Origin of Life><Outbreaks><Pathway interactions><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Poxviridae><Poxviridae Infections><Poxviridae disease><Poxvirus Infections><Poxviruses><Process><Production><Protein Phosphorylation><Proteins><Pyruvate><RAFT1><RNA Expression><Recording of previous events><Regulatory Protein><Repression><Research><Research Proposals><Role><Signal Transduction><Signal Transduction Systems><Signaling><Site><Smallpox><Structural Protein><TCA cycle><Testing><Therapeutic><Transcription><Transphosphorylases><Tricarboxylic Acid Cycle><Variola><Viral><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virus><Virus Replication><Wart-Hog Disease Virus><Zoonoses><Zoonotic><Zoonotic Infection><aerobic glycolysis><antagonism><antagonist><balance><balance function><biological signal transduction><cell biology><cell type><cryo-EM><cryoEM><cryogenic electron microscopy><domain mapping><fatty acid oxidation><genetic regulatory protein><histories><host response><immune evasive><immune system response><immunoresponse><insight><mRNA><mRNA Translation><mTOR><mammalian target of rapamycin><member><mitochondrial><mpox><mpox virus><mpxv><mtDNA><mutant><novel><pandemic concern><pandemic potential><pandemic risk><pandemic threat><pathway><plasmid vaccine><pox virus><prevent><preventing><protein function><regulatory gene product><response><screening><screenings><sensor><small pox><small pox eradication><smallpox eradication><social role><variola major><vector vaccine><viral DNA><viral detection><viral multiplication><viral replication><virus DNA><virus detection><virus multiplication><virus protein>