TFAM in corneal inflammation and infection - Project Summary: Corneal inflammation–induced blindness is a major global health burden, with herpes simplex virus-1 (HSV-1) being a leading cause due to its ability to establish recurrent infections and trigger chronic inflammation. HSV-1 evades host immunity by disrupting mitochondrial quality control pathways, particularly the clearance of mitochondrial DNA (mtDNA), a key activator of the cGAS–STING–IRF3/7 axis and type I interferon (IFN-I) responses. Our preliminary data, supported by prior studies, reveal that HSV-1 induces mitochondrial stress and promotes the early degradation of mtDNA and its packaging protein TFAM (mitochondrial transcription factor A) through nucleophagy, a selective autophagy process. Under homeostatic conditions, mitochondrial DNA (mtDNA) is sequestered within the mitochondrial matrix. However, cellular stressors including oxidative damage, mitochondrial membrane permeabilization, and defective mitophagy, can trigger mtDNA release into the cytosol, particularly during viral infection. Once in the cytosol, mtDNA serves as a potent danger-associated molecular pattern (DAMP), activating innate immune sensors, triggering a robust type I interferon (IFN-I) response, and establishing an antiviral state within the cell. We propose that the viral protein UL12.5 acts as a molecular trigger to promote mtDNA release into the cytosol, while TFAM mediates the degradation of this cytosolic mtDNA via autophagy. This process requires the autophagy adaptor OPTN, which facilitates TFAM-dependent nucleophagy. Disruption of autophagy or loss of OPTN leads to mtDNA retention, cGAS–STING activation, enhanced IFN-I signaling, and exacerbated corneal pathology. Importantly, aging impairs mitochondrial function and autophagic flux, potentially amplifying these defects and increasing susceptibility to HSV-1–induced hyperinflammatory disease. During the K99 phase, we will investigate how TFAM, UL12.5, and OPTN regulate mtDNA clearance and antiviral signaling using CRISPR-Cas9-generated knockout HCE cells, autophagy-deficient models, and OPTN knockout mice. We will assess infection-induced mitochondrial dysfunction, type I interferon (IFN-I) responses, and corneal pathology. In the R00 phase, we will evaluate how aging impacts TFAM-mediated nucleophagy and antiviral responses in aged mice and validate our findings using autophagy, TFAM, and STING-deficient models. This project will dissect how HSV-1 hijacks TFAM-mediated nucleophagy to degrade mtDNA and evade immune detection. Specifically, we will (1) define TFAM’s role in mtDNA degradation during HSV-1 infection, (2) examine UL12.5-mediated mtDNA release, (3) determine OPTN’s role as a TFAM adaptor in nucleophagy, and (4) assess how aging and host genetics shape these processes. Together, these studies will uncover a novel immune evasion strategy by HSV-1 and establish TFAM-mediated nucleophagy as a critical regulator of mitochondrial immunity, and corneal inflammation, informing host-directed antiviral strategies for vulnerable populations.
