Childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common viral encephalitis in Western countries. The pathogenesis of HSE had long remained unclear. Over the last decade, we have discovered that forebrain HSE may result from mono-allelic or bi-allelic single-gene mutations impairing TLR3-, IFN-a/b- mediated immunity to HSV-1 in the central nervous system (CNS) in some children, whereas brainstem HSE may result from inborn errors of RNA lariat metabolism due to bi-allelic mutations in DBR1. We also demonstrated that patient-specific induced pluripotent stem cells (iPSC)-derived TLR3-deficient cortical neurons and oligodendrocytes were highly susceptible to HSV-1 infection, suggesting that impaired TLR3-IFN-mediated CNS intrinsic anti-HSV-1 immunity underlies the pathogenesis of forebrain HSE in patients with inborn errors of TLR3 immunity. While testing a hypothesis that HSE in other children may result from a collection of unknown single-gene inborn errors of immunity against HSV-1, we surprisingly discovered four mono-allelic mutations in SNORA31 in five unrelated forebrain HSE patients, out of the 205 HSE children studied by whole exome sequencing (WES). SNORA31 encodes a small noncoding RNA (snoRNA) of the H/ACA class, snoRNA31, which is predicted to act as a guide RNA directing the chemical modification of target uridine residues to pseudouridine, at position 218 of the 18S rRNA and position 3,713 of the 28S rRNA. How heterozygous mutations in SNORA31 could underlie HSE pathogenesis is completely unclear.
We aim to test a hypothesis that human snoRNA31 defines a new, CNS-specific mechanism of intrinsic anti-HSV-1 immunity, at both the molecular and cellular levels. Our preliminary results showed that dermal fibroblasts from four SNORA31- mutated patients display enhanced HSV-1 susceptibility, same as SNORA31-mutated cortical neurons differentiated from patient-specific iPSCs. We will analyze the specific role of snoRNA31 in CNS-intrinsic antiviral immunity and its underlying mechanisms following a hypothesis-testing approach and a hypothesis-generating approach, taking advantage of next generation sequencing, iPSC reprogramming, CRISPR/Cas9, and neuronal cell differentiation technologies. In the hypothesis-generating approach, we will investigate the antiviral activity of snoRNA31, indirectly via TLR3- or IFN-mediated immunity, or directly via the suppression of viral replication, in in vitro assays. We will also investigate HSV-1 susceptibility in various SNORA31-mutated cell types including iPSC-derived CNS cortical neurons. In the hypothesis-driven approach, we will perform a genome-wide host cellular and viral transcriptome analysis in SNORA31-mutated or WT iPSC-derived cortical neurons upon HSV- 1 infection or stimulation with poly(I:C) or IFN-?, in order to unravel any abnormally regulated host immune pathway(s), and any abnormally expressed key host protein-coding RNA, ncRNA or viral RNA, in SNORA31- mutated cells, for further experimental characterization. This research will unravel a novel molecular and cellular mechanism of HSE pathogenesis, which will open new therapeutic avenues.

Public Health Relevance

Childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Single-gene mutations impairing TLR3-dependent, IFN-a/b-mediated immunity to HSV-1 in the central nervous system (CNS) underlie the pathogenesis of forebrain HSE in a small proportion of affected children, whereas mutations in DBR1 impairing RNA lariat metabolism and cell-intrinsic immunity to HSV-1 underlie the pathogenesis of brainstem HSE in some of the other children. We propose to study a novel CNS- specific mechanism of cell-intrinsic antiviral immunity against HSV-1, which may, but not necessarily be related to the TLR3-IFN circuit, in forebrain HSE children with mutations in SNORA31.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Clinical Neuroimmunology and Brain Tumors Study Section (CNBT)
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Beisel, Christopher E
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Rockefeller University
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New York
United States
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