The role of mTOR dysregulation in poxvirus infection - 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.
