Characterization of a sensor domain for cytoplasmic nucleic acid in the antiviral factor, SAMD9

Project Summary Monkeypox virus (mpox) is an emerging zoonosis with an oropharynx route of transmission that causes oral pathology in approximately 70% of infected individuals. Studies suggest that transmissibility of mpox has increased, and with the waning of poxvirus immunity in younger generations, there is a need to understand host immune responses against mpox. The closely related vaccinia virus (VACV) and mpox can enter nearly all cell types and replicate exclusively in the cytoplasm, making intracellular factors critical host barriers. Sterile Alpha Motif Domain-containing 9 (SAMD9) is a self-regulating cytosolic host restriction factor for poxviruses. SAMD9 is ubiquitously expressed across several cell types within the oral mucosa, making it a host barrier for oral mpox transmission. Understanding the mechanism of how SAMD9 regulates mpox replication is essential in developing effective antiviral strategies to mitigate oral pathology in infected individuals. While SAMD9 is predicted to function like a pathogen recognition receptor (PRR), the mechanism by which SAMD9 senses viral infection and is activated to mitigate viral replication remains unknown. SAMD9 is predicted to have a TPR/OB domain that serves as a sensor domain. The early phase of VACV infection, during which double-stranded DNA enters the cytoplasm, is sufficient for SAMD9 activation. This proposal addresses the hypothesis that the TPR/OB is a sensor domain that recognizes cytosolic nucleic acid to induce SAMD9 activation. The physiological and structural basis for SAMD9 activation will be investigated using VACV as a model for mpox infection. First, antiviral experiments will be conducted to pinpoint the stage of infection during which SAMD9 is activated. This will be verified with in vitro biochemical studies that test what nucleic acid type activates SAMD9 outside the context of infection. Next, biochemical experiments using recombinant TPR/OB protein will determine what type of nucleic acid binds to this domain, and the physiological basis for this binding activity will be investigated with antiviral assays and in vitro assays. This proposal seeks to elucidate the danger signals and sensor domain involved in activating SAMD9 to induce its antiviral activity. The proposed work is significant, as it will uncover the mechanism behind how SAMD9 can protect against viral infection and identify the stage of poxvirus infection during which it is activated. The proposal has the potential impact of helping to develop effective antiviral strategies against future mpox outbreaks and will open new areas of investigation between SAMD9 and other viruses of the oral cavity. This project is innovative in that it will help to establish SAMD9 as a novel pathogen recognition receptor for cytosolic nucleic acid. Completion of the proposed work will provide me with the necessary knowledge and technical skills to become an independent investigator in the field of virus-host interactions.