A cellular examination of TLR3 antiviral function in human iPSC-derived CNS and sensory neurons

Ewaleifoh, Osefame

doi:https://doi.org/10.21985/n2-3h4x-nt24

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A cellular examination of TLR3 antiviral function in human iPSC-derived CNS and sensory neurons

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Herpes simplex encephalitis (HSE) is a rare and severely debilitating life-threatening disease caused by herpes simplex virus type 1 (HSV-1) infection of the human central nervous system (CNS). The severe and often fatal outcomes of HSE contrast starkly with the more frequent and largely benign consequences of herpes labialis. Whole exome sequencing of a cohort of HSE patients identified several inborn genetic mutations, each of which increased the susceptibility of patient derived fibroblast to HSV-1 infection in vitro. Innate immune signaling proteins with inborn genetic mutations identified in HSE patients to date include: signal transducer and activator of transcription 1 (STAT1), NF-κB essential modulator (NEMO), toll-like receptor 3 (TLR3), uncoordinated 93 homologue B (Unc93B), TIR-domain-containing adaptor-inducing interferon-β (TRIF) and an RNA lariat debranching enzyme 1 (Dbr1). More mutations are yet to be discovered and characterized. All together over 20 HSE patients presenting focal inflammation of the frontal/temporal lobes of the brain with genetic errors in TLR3 or TLR3-accessory proteins have been reported. However, despite the increased number of genetic mutations associated with HSE, our understanding of how these mutations contribute to HSE or in vitro susceptibility to HSV-1 infection remains limited. To advance our understanding of how genetic mutations contribute to in vitro susceptibility to HSV-1 infection, in this thesis I investigate the antiviral mechanism of TLR3 in human iPSC-derived neurons. Previous in vitro studies with fibroblasts and iPSC-derived CNS neurons from HSE patients established that cells from HSE patients with TLR3-associated mutations display increased susceptibility to HSV-1. In this thesis, I employed several fluorescent reporter viruses coupled with live-cell fluorescence microscopy to monitor the progression of HSV-1 infection in human iPSC-derived neurons. I demonstrate that human cortical neurons evoke an unusual response to HSV-1 that immediately blocks the fusion of viral particles into the cells. In contrast, HSV-1 entry and infection occurred unabated in cortical neurons from TLR3-deficient patients. The TLR3-dependent early block to HSV-1 infection was absent from iPSC-derived sensory neurons, which otherwise retained a functional and inducible antiviral response to HSV-1 infection. The early block to HSV-1 infection observed in iPSC-derived CNS neurons was independent of STAT1, but inhibited by antagonists of MLKL and RIPK3. In support of the latter, this research lead to the identification of a HSE patient with inborn errors in RIPK3 whose primary fibroblasts displayed enhanced susceptibility to HSV-1 infection following in vitro infection. Finally, purified RNAse-treated HSV-1 failed to trigger TLR3-dependent antiviral signaling and evaded the constitutive resistance of iPSC-derived CNS neurons. Taken together, this work reveals a previously unreported cell-type-dependent rapid non-canonical TLR3 antiviral response that restricts HSV-1 entry into human iPSC-derived cortical neurons, and raises a cautionary note that inhibitors of RIPK3 and MLKL currently in clinical use may increase a patient’s risk for HSE.

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