Long double-stranded RNA (dsRNA) is a hallmark of viral replication and a prototypical pathogen-associated molecular pattern, yet it remains difficult to sequence because of its relative rarity, thermostability and poor compatibility with conventional sequencing workflows. We introduce Size-Accurate Long Double-stranded RNA-Sequencing (SALDOR-Seq), a dual-selection approach that combines stringent nuclease-based depletion of single-stranded RNA with adapter ligation and single-primer amplification to selectively recover intact dsRNA and preserve native length. SALDOR-Seq achieves high specificity and ~15,000-fold higher dsRNA recovery than short-read workflows, enabling full-length sequencing of viral dsRNA genomes exceeding six kilobases. Applied to RNA from virus-infected cells, it resolved replication-associated dsRNA intermediates, identified A-to-I editing within cytomegalovirus-derived dsRNA, and, through metagenomic analysis, revealed occult mycovirus infection in Aspergillus fumigatus. SALDOR-Seq therefore provides a dedicated, length-preserving method for high-confidence dsRNA profiling in complex samples and expands experimental access to dsRNA relevant for the study of viral replication, innate immunity and pathogen detection.

Abstract Human respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants. However, host factors that influence disease severity remain incompletely defined. While clinical risk factors are known, identifying genetic susceptibility has been challenging. In this study, we combined human genetics with functional virology to identify host factors that modulate RSV infection and spread. Starting from a cohort of infants hospitalized with severe RSV disease, we prioritized rare coding variants present in homozygous form and predicted to cause strong functional impairment, and selected candidate genes for mechanistic follow-up. Functional interrogation of 23 candidates by CRISPR/Cas9 knockout screening in human lung epithelial cells identified unconventional myosin-X (MYO10), encoding the actin-based motor protein myosin-X, as a critical host factor for RSV. Genetic disruption or siRNA-mediated depletion of MYO10 significantly reduced RSV infectivity, with the strongest effects at post-entry stages of the viral life cycle. Loss of MYO10 impaired filopodia formation, cell migration, and wound healing, leading to altered cell–cell connectivity and restricted viral dissemination. MYO10 depletion reduced both short-range cell-to-cell transmission and longer-distance extracellular spread, resulting in fewer infected cells and diminished accumulation of progeny virus in culture supernatants. In contrast, RSV entry, early gene expression, and interferon responses were unaffected. Finally, a rare homozygous MYO10 motor-domain variant (rs7737765; H148Y), enriched in severe cases, also reduced RSV replication in cell culture—opposite to expectations for a risk allele—yet underscoring biological relevance and suggesting that MYO10 variation may influence disease in vivo through additional effects on epithelial function. Importance Respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in infants, yet it remains unclear why some children develop more serious disease than others. In this study, we combined patient genetic data with laboratory experiments to identify host factors that influence how RSV spreads in the lung. We found that the human protein MYO10, which mediates the formation of small cell protrusions, plays a key role in enabling the virus to spread from cell to cell. When MYO10 was disrupted, viral spread was strongly reduced, even though early steps of infection were unaffected. Interestingly, a rare genetic variant in MYO10 found in patients also altered viral replication, highlighting potential clinical relevance. These findings provide new insight into how host cell architecture contributes to RSV infection and suggest that targeting host pathways involved in viral spread could complement existing antiviral strategies.

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutières syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn²⁺-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a key post-transcriptional modification that regulates RNA splicing, stability, and translation. Dysregulation of ADAR activity caused by mutations in ADAR1 leads to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder characterized by aberrant activation of Melanoma differentiation-associated protein 5 (MDA5) by self RNA and excessive type I interferon production. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Here, we show that ADAR1 protein levels and RNA editing activity in mammalian cells critically depend on the cofactor inositol hexakisphosphate (IP6). Using Inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, next-generation sequencing (NGS), and a cell-permeable IP6 prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. In vitro ADAR1 translation and RNA editing assays revealed that IP6 contributes to the folding and full catalytic activity of ADAR1, and that inositol pentakisphosphate (1,3,4,5,6-IP5) can partially substitute IP6 as a cofactor. Molecular dynamics simulations and biochemical analyses identified the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly governs Zn2+-ion positioning through interactions with key residues, including K1039 and N907. Notably, the AGS-associated N907S mutation impairs RNA editing, by altering IP6 coordination and introducing a more dynamic situation in the hydrogen-bonding network that linked IP6 and Zn2+-ion. Together, these findings identifies IP6 as an essential cofactor and regulator of ADAR1 activity and highlights cofactor availability and interaction networks as potential strategies for therapeutically modulating RNA editing in disease. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, Project-ID 369799452— TRR237 - A02, Project-ID 548714673, CIBSS – EXC-2189 – Project ID 390939984 Volkswagen Foundation, VW Momentum Grant 98604 Israel Science Foundation, 2637/23 Medical Research Council, MR/T028904/1

The innate immune system is known for its ability to recognize cytosolic DNA as evidence of infection, but detailed studies of this process have been mostly limited to mice and cell lines. To investigate inflammasome responses in human primary cells, we used engineered viruses encoding the inflammasome reporter caspase-1CARD-EGFP. We show that released genomes of vaccinia virus and monkeypox virus trigger robust inflammasome assembly in human primary cells. To determine the involved inflammasome sensors, we generated nanobodies against AIM2. Three of them inhibit AIM2 inflammasome assembly by blocking the polymerization of the AIM2 Pyrin domain, most potently as bivalent nanobodies. Utilizing an engineered vaccinia virus expressing bivalent AIM2 nanobodies, we demonstrate that inflammasomes in primary human macrophages and keratinocytes are nucleated by AIM2, while CD14+ monocytes assemble NLRP3 inflammasomes. This finding resolves the discrepancy between the previously reported activation of AIM2 inflammasomes in mice and NLRP3 inflammasomes in humans, and provides the first evidence for cell-type-specific regulation of DNA-triggered inflammasome activation. The newly developed AIM2-specific nanobodies offer a precise tool to dissect and potentially target AIM2 inflammasome assembly in other disease contexts.

Photochemical control of oligonucleotides bears great potential for the spatio‐temporal control of therapeutic targets, such as immune sensing receptors. Retinoic acid‐inducible gene I (RIG‐I) is a cytoplasmic receptor of the innate immune system that triggers antiviral responses upon detection of viral RNA.RIG‐I can be specifically activated by short double‐stranded (ds) RNA with a blunt 5′ end bearing a triphosphate, mimicking nascent viral transcripts. Tumor cells are specifically sensitive to RIG‐I‐induced cell death. Here we developed a potent oligonucleotide ligand for spatiotemporally controlled activation of RIG‐I by light exposure. Through structural considerations and functional studies we identified a combination of two nucleoside positions in a RIG‐I oligonucleotide ligand for which the substitution of both respective 2′‐hydroxy groups of the ribose by photolabile protecting groups (2′‐photocages) resulted in a complete loss of RIG‐I ligand activity, whereas photocaging the individual positions was not sufficient to turn off RIG‐I. Light exposure fully restored RIG‐I activation by the photocaged RIG‐I ligand, enabling light‐controlled RIG‐I‐mediated cell death of human cancer cells which had internalized the photocaged RIG‐I ligand prior to light exposure. This novel photoactivatable RIG‐I oligonucleotide ligand may be applicable for precise light‐controlled induction of tumor cell death in superficial cancer such as melanoma.

Die photochemische Kontrolle von Oligonukleotiden hält großes Potenzial für die spatiotemporale Kontrolle therapeutischer Zielstrukturen wie etwa Immunrezeptoren. Retinsäure‐induzierbares‐Gen‐I (RIG‐I) ist ein zytosolischer Rezeptor des angeborenen Immunsystems, der bei Erkennung viraler RNA antivirale Immunantworten auslöst. RIG‐I kann gezielt durch kurze doppelsträngige (ds) RNA mit einem glatten, Triphosphat (ppp)‐tragenden 5′‐Ende aktiviert werden, die naszierende virale Transkripte imitiert. Tumorzellen sind besonders empfindlich gegenüber RIG‐I‐induziertem Zelltod. In dieser Arbeit entwickelten wir einen potenten Oligonukleotid‐Liganden zur spatiotemporal kontrollierten RIG‐I‐Aktivierung durch Licht. Mit Hilfe struktureller Überlegungen und funktioneller Studien identifizierten wir eine Kombination von zwei Nukleosidpositionen in einem RIG‐I‐Oligonukleotid‐Liganden, an denen der Austausch der jeweiligen 2′‐Hydroxygruppen der Ribose durch photolabile Schutzgruppen (2′‐Photocages) zu einem vollständigen Verlust der RIG‐I‐Ligandenaktivität führte. Der Austausch an nur einer einzelnen Position reichte hingegen nicht aus, um die RIG‐I‐Aktivierung vollständig zu verhindern. Lichtbestrahlung stellte die RIG‐I‐Aktivierung des photogeschützten RIG‐I‐Liganden vollständig wieder her und ermöglichte einen lichtkontrollierten, RIG‐I‐vermittelten Zelltod in humanen Krebszellen, die zuvor den lichtaktivierbaren RIG‐I‐Liganden aufgenommen hatten. Dieser neuartige lichtaktivierbare RIG‐I‐Oligonukleotidligand könnte zur präzisen lichtkontrollierten Induktion des Tumorzelltods bei oberflächlichen Krebserkrankungen, wie Melanomen anwendbar sein.

Introduction Inflammation, induced by proinflammatory cytokines and other mediators, plays an important role in the initiation of immunological response. Immune cells must adapt to their tissue environment to provide appropriate responses. Adenoid tissue is part of the Nasopharynx-Associated Lymphoid Tissue (NALT) and functions as a first line immunological defense against pathogens entering the body from the outside. Little is known about how the innate immune response is regulated within the adenoid tissue.

CD163+ TAMs are associated with lymphogenesis. A, LVD and LVI were assessed in the stroma using D2-40–stained lymphatic endothelium. Intratumoral and stromal CD163 immunoreactivity was recorded as in Fig. 1B; data are presented as mean immunoreactivity normalized to tissue compartments in percent. CD163+ TAMs were categorized on the basis of high/low LVD and the presence/absence of LVI. Individual datapoints, shown as dots, overlap summary statistics boxplots with medians represented by horizontal center lines. Significance analysis by two-sided Mann–Whitney U test with Benjamini–Hochberg procedure. B, BVD and BVI were assessed in the stroma using CD31-stained vessels. Intratumoral and stromal CD163 immunoreactivity was recorded as in Fig. 1B; data are presented as mean immunoreactivity normalized to tissue compartments in percent. CD163+ TAMs were categorized on the basis of high/low BVD and the presence/absence of BVI. Individual datapoints, shown as dots, overlap summary statistics boxplots with medians represented by horizontal center lines. Significance analysis by two-sided Mann–Whitney U test with Benjamini–Hochberg procedure.

Supplementary Table 3 summarizes clinicopathological characteristics of the discovery cohort.

Abstract The incidence rates of vulvar squamous cell cancer (VSCC) have increased over the past decades, requiring personalized oncologic approaches. Currently, lymph node involvement is a key factor in determining prognosis and treatment options. However, there is a need for additional immune-related biomarkers to provide more precise treatment and prognostic information. Here, we used IHC and expression data to characterize immune cells and their spatial distribution in VSCC. Hierarchical clustering analysis identified distinct immune subtypes, of which the macrophage-rich subtype was associated with adverse outcome. This is consistent with our findings of increased lymphogenesis, lymphatic invasion, and lymph node involvement associated with high macrophage infiltration. Further in vitro studies showed that VSCC-associated macrophages expressed VEGF-A and subsequently induced VEGF-A in the VSCC cell line A-431, providing experimental support for a pro-lymphangiogenic role of macrophages in VSCC. Taken together, immune profiling in VSCC revealed tumor processes, identified a subset of patients with adverse outcome, and provided a valuable biomarker for risk stratification and therapeutic decision making for anti-VEGF treatment, ultimately contributing to the advancement of precision medicine in VSCC. Significance: Immunoprofiling in VSCC reveals subtypes with distinct clinical and biological behavior. Of these, the macrophage-rich VSCC subtype is characterized by poor clinical outcome and increased VEGF-A expression, providing a biomarker for risk stratification and therapeutic sensitivity.

Supplementary Figure 3 shows expression of VEGF proteins.

Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.

VEGF-A expression by in vitro polarized TAMs. A, Healthy donor monocytes were differentiated into macrophages using GMCSF and exposed to different culture conditions: medium alone (monocyte-derived macrophage) or A-431 supernatant (in vitro polarized TAM). Immunofluorescent staining was performed on day 5 and compared with A-431 cells. Cells were stained with anti-VEGF-A (clone VG-1; cytoplasma, green), PKH26 (cell membrane, red), and Hoechst 34580 (cell nucleus, blue). Representative overlay figures are depicted (32x magnification); white scale bar length 200 µm. B, Macrophages were generated as in A and intracellular VEGF-A expression was determined by flow cytometry (anti-VEGF-A clone 23410) on day 5. Data combine results from two independently analyzed individuals. Data are given as d-gMFIs. Individual datapoints, shown as dots, overlap summary statistics barplots (mean ± SEM). Significance analysis by two-sided Student t test. C, Macrophages were generated as in A, and in vitro polarized TAMs and A-431 cells were cultured alone (monoculture) or together (coculture with 2:1 ratio) in the presence of LPS. Intracellular VEGF-A expression was determined by flow cytometry using two anti-VEGF-A antibody clones as indicated after 24 hours of coculture following a 4-hour restimulation with Brefeldin A. For comparison between different cell types, specific cell populations of interest were identified on the basis of their expression of phenotypic markers (EpCAM for cancer cells, CD14 for macrophages), a negative control was included, and cocultured cells were normalized to VEGF expression of blood lymphocytes. Data combine results from three individuals and are given as d-gMFIs. Individual datapoints, shown as dots, overlap summary statistics boxplots with medians represented by horizontal center lines. Significance analysis by two-sided Student t test.

Density of stromal TAMs affect outcome. Survival analysis of intratumoral and stromal immune cell counts

Supplementary Figure 2 shows analysis in independent validation cohorts.

The TAMhigh immune cell cluster is associated with poor outcome. A, Heat map shows the distribution of immune cells (intratumoral and stromal combined). Immunoreactivity was recorded as in Fig. 1B. Unsupervised clustering of log2-transformed cell count data from 41 samples was performed using Euclidean distance. B, Kaplan–Meier plots depict OS and RFS of patients stratified by TAM clusters from A; significance analysis by log-rank test.

Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the 'gold standard' small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.