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.