Type I and type III interferons (IFNs) are induced by only a small fraction of virus-infected cells, and pre-existing (intrinsic, IFN-independent) expression of the interferon-stimulated gene (ISG) OASL was recently shown to license IFN induction during influenza A virus infection. What remains unresolved is the mechanistic basis of this heterogeneity: which regulatory mechanisms make some cells intrinsically ISG-positive at steady state, how these features vary across ISGs, cell types, tissues, and individuals, and what the resulting frequency of IFN-competent responder cells means for susceptibility to viral infection and for IFN-driven autoimmune (interferonopathy) disease. A same-group follow-up identified tonic (low-level, continuous) JNK/AP-1 signaling state — JNK being the c-Jun N-terminal kinase stress-signaling pathway and AP-1 the activator protein-1 transcription factor family it controls — as one determinant in a transformed cell line, but the mechanisms, their primary-cell and in-vivo validation, and the disease consequences remain open.

Background

Type I (IFN-α/β) and type III (IFN-λ) interferons are among the earliest antiviral defenses at mucosal surfaces. Their induction begins when pattern-recognition receptors such as RIG-I (retinoic acid-inducible gene I, a cytosolic RNA helicase that recognizes viral RNA) activate IRF (interferon regulatory factor) transcription factors to switch on IFN genes. IFN then acts in paracrine and autocrine fashion through JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling to upregulate interferon-stimulated genes (ISGs), a large program encoding antiviral effectors and signaling amplifiers. A long-standing observation is that this response is strikingly heterogeneous at the single-cell level: in influenza A virus (IAV)-infected epithelial cultures, only a small minority of infected cells produce detectable IFN, and single-cell studies have attributed part of this heterogeneity to stochastic IFNB1 promoter activation and to cell-to-cell variation in the abundance of sensors and IRF transcription factors at the time of stimulation. OASL (2'-5'-oligoadenylate synthetase-like protein) is an ISG whose antiviral activity is not enzymatic: human OASL binds double-stranded RNA and enhances RIG-I signaling by promoting RIG-I oligomerization, circumventing the usual requirement for K63-linked polyubiquitination of the RIG-I CARD (caspase activation and recruitment) domains. OASL is also known to function in neuroinflammation contexts, and its expression is normally thought of as IFN-induced. Separately, an IFN-independent layer of antiviral defense has been documented in specific cell types: stem cells maintain constitutive (intrinsic) ISG expression that protects them from viral infection, and basal expression of IRF1 in hepatocytes drives constitutive ISG transcription and intrinsic viral resistance. Whether and how such intrinsic ISG expression varies within differentiated cell populations, and what it does there, was unclear. Rivera-Cardona et al. addressed this by developing a pseudotime-reconstruction method (computational ordering of single-cell transcriptomes along the infection time course) for temporal single-cell RNA sequencing (scRNA-seq, transcriptome-wide profiling of individual cells) data of IAV-infected A549 cells (a widely used human lung adenocarcinoma-derived epithelial cell line), which identified several intrinsically expressed ISGs (including OASL) in pre-infection cells as correlates of a cell's later IFN induction potential; validation experiments confirmed that intrinsic OASL expression is required for robust IFNL1 (the gene encoding the type III IFN IFN-λ1) induction during IAV infection, acting as a license for RIG-I-mediated signaling in a subset of 'IFN-competent sentinel' cells. In this model the frequency of responder cells within a tissue sets how far viral replication must proceed before an IFN response is triggered. The published Discussion states explicitly that the mechanisms governing the intrinsic expression patterns of OASL and other ISGs across cell populations at steady state remain an open question, notes preliminary evidence for substantial person-to-person and cell-type variation in intrinsic ISG expression in primary human airway cells (relatively high in ciliated cells, and in basal/cycling basal cells, possibly related to a less-differentiated state), and raises the question of whether individuals with elevated frequencies of intrinsic OASL-expressing cells are more prone to inappropriate IFN responses to self-RNA — a concern anchored in the type I interferonopathies, monogenic autoinflammatory disorders such as Aicardi-Goutières syndrome in which gain-of-function mutations that lower RIG-I-like receptor activation thresholds cause recognition of self-RNAs. A related adjacent literature on tonic (low-level, continuous) IFN signaling — shown to vary between immune cell types in mice and between healthy humans, and to modulate susceptibility to SARS-CoV-2 in human lung — documents individual- and cell-type-level variation in IFN system tone, but concerns IFN-dependent signaling rather than the IFN-independent intrinsic ISG expression identified here.

Problem Statement

Identify the mechanism(s) — genetic, epigenetic, and signaling — that govern IFN-independent intrinsic expression of OASL and other interferon-stimulated genes in individual cells at steady state, and establish how these regulatory features vary across different ISGs, cell types, tissues, and individuals, including the mechanistic basis of the observed heritability of IFN-induction potential (the stable inheritance of that potential across cell generations, not genetic heritability). Then determine the functional consequences of the resulting cell-to-cell heterogeneity: to what extent the frequency of intrinsically ISG-positive, IFN-competent responder cells predicts the outcome of viral infection in primary cells and in vivo, whether elevated frequencies of intrinsic OASL-expressing cells promote inappropriate IFN responses to self-RNA and thereby increase interferonopathy (e.g., Aicardi-Goutières-spectrum) risk in the absence of infection, and how intrinsic ISG expression balances host defense against immunopathology across diverse cell types and tissues. Answers must go beyond correlative single-cell associations in transformed cell lines: the proposed regulators of intrinsic ISG expression require causal validation (gain- and loss-of-function perturbation with single-cell readouts of steady-state ISG expression and subsequent IFN induction potential, with IFN-independence established under JAK/IFN-receptor blockade) and demonstration in primary cells or in vivo systems. A causal mechanistic identification of one or more regulators of IFN-independent intrinsic ISG expression that meets these requirements is the central mechanistic question; the characterization of variation across ISGs, cell types, tissues, and individuals, the prediction of infection outcome, and the interferonopathy-consequence arms are independent sub-questions.

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