UC Irvine

Alpha herpesviruses (αHVs), including human pathogens, herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), as well as the animal pathogen, pseudorabies virus (PRV), initially infect mucosal epithelial cells. Subsequently, they invade peripheral neurons, establishing lifelong latent infections in the peripheral nervous system (PNS). While mucosal epithelia antiviral defenses are well characterized, the mechanisms governing neuronal antiviral responses remain poorly understood. Due to their postmitotic status and cellular polarity, neurons depend on antiviral responses that do not provoke inflammatory damage. Despite this constraint, neurons are exposed to a complex cytokine milieu produced by infected mucosal epithelia. Type III interferons (e.g IFN lambda; IFN-λ) are among the most abundant cytokines secreted by infected mucosa. This dissertation research elucidates mechanisms of how neurons respond to IFN-λ signaling, identifies key neuronal antiviral effectors, and reveals how αHVs, specifically HSV-1, evade these IFN-λ-mediated defenses during infection. We first characterized IFN-λ signaling in PNS neurons and found that, unlike canonical IFN signaling, IFN-λ treatment specifically activated phosphorylation of STAT1 (but not STAT2) in neurons. Transcriptomic profiling revealed biphasic kinetics: an early phase associated with neuronal remodeling and a later phase characterized by the upregulation of interferon-stimulated genes (ISGs). RSAD2 (i.e. viperin) emerged as the most strongly upregulated effector during late-stage IFN-λ treatment, localizing to perinuclear ER membranes in a neuron-specific manner. Other canonical ISGs of interest included Mx1, a GTPase that inhibits viral replication, and Usp18, an enzyme that de-conjugates ISG15 to fine-tune and negatively regulate IFN signaling- demonstrating that neurons mount antiviral responses to IFN-λ while exhibiting unique temporal and spatial characteristics tailored to their post-mitotic nature. Functional studies demonstrated that IFN-λ pre-treatment restricts pseudorabies virus (PRV) replication in both fibroblasts and primary neurons, with pre-treatment prior to infection. In contrast, post-infection treatment yielded only modest inhibition, indicating that once immediate-early and early viral proteins accumulate, IFN-λ signaling becomes more restricted. Notably, antiviral restriction was retained even at higher MOIs, suggesting that neurons can sustain an antiviral program sufficiently strong to control viral replication even under high inoculum pressure. These findings reveal that IFN-λ produced at mucosal surfaces can establish a preemptive antiviral state in innervating sensory neurons, creating a barrier that limits or delays neuroinvasion. Strikingly, while PRV was highly sensitive to IFN-λ restriction, HSV-1, exhibited resistance. This resistance is conferred by the neurovirulence factor ICP34.5, a gene uniquely encoded by the simplexvirus genus (HSV-1 and HSV-2) but absent in the varicelloviruses (VZV and PRV). Using ICP34.5-deficient HSV-1 mutants, we demonstrated that ICP34.5 counteracts the neuronal IFN-λ- RSAD2-mediated response through two mechanisms: (1) reversing eIF2α phosphorylation via PP1α recruitment, and (2) disrupting RSAD2 localization at the perinuclear membrane. In separate studies on the characterization of RSAD2, we uncover a mucosal-PNS antiviral axis involving IFN-λ-driven responses and RSAD2-dependent restriction of αHV (varicellovirus) neuroinvasion, which simplexviruses expressing ICP34.5 can bypass. These results highlight the divergent neuronal adaptation of two different αHV genera displaying different immune evasion strategies and identifies RSAD2 as a neuron-specific antiviral target with therapeutic implications for preventing neuroinvasion. These discoveries led to the investigation of spatially distinct IFN-λ-mediated antiviral defenses in neuronal axons. Local axonal STAT1 activation was observed, which reduced retrograde αHV transport in axons, establishing an early immune checkpoint prior to viral genome delivery to the nucleus. Priming axons with IFN-λ decreases virion movement towards the cell body and delays PRV infection at later stages of infection in neuronal cell bodies. These findings position the axonal compartment as a promising therapeutic target and suggest that strategies that deliver IFN-λ to peripheral nerve termini or recruit activated STAT1 in axons during early infection could prevent viral neuroinvasion and may affect the establishment of latency in the PNS. Although further studies are required to elucidate the mechanism of this non-canonical IFN-STAT signaling in axons, these findings fundamentally advance our understanding of neuronal immune responses and axonal responses against neuroinvasion.