Impaired STING Activation Due to a Variant in the E3 Ubiquitin Ligase AMFR in a Patient with Severe VZV Infection and Hemophagocytic Lymphohistiocytosis.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor (AMFR), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-ß reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

CDK1 couples proliferation with protein synthesis.

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection.

UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen, and about one third of the global population will be exposed to the virus in their lifetime. HBV infects hepatocytes, where it replicates its DNA and infection can lead to acute and chronic hepatitis with a high risk of liver cirrhosis and hepatocellular carcinoma. Despite this, there is limited understanding of how HBV establishes chronic infections. In recent years it has emerged that foreign DNA potently stimulates the innate immune response, particularly type 1 interferon (IFN) production; and this occurs through a pathway dependent on the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase and the downstream adaptor protein stimulator of IFN genes (STING). In this work we describe that human and murine hepatocytes do not express STING. Consequently, hepatocytes do not produce type 1 IFN in response to foreign DNA or HBV infection and mice lacking STING or cyclic guanosine monophosphate-adenosine monophosphate synthase exhibit unaltered ability to control infection in an adenovirus-HBV model. Stimulation of IFN production in the murine liver by administration of synthetic RNA decreases virus infection, thus demonstrating that IFN possesses anti-HBV activity in the liver. Importantly, introduction of STING expression specifically in hepatocytes reconstitutes the DNA sensing pathway, which leads to improved control of HBV in vivo. CONCLUSION: The lack of a functional innate DNA-sensing pathway in hepatocytes hampers efficient innate control of HBV infection; this may explain why HBV has adapted to specifically replicate in hepatocytes and could contribute to the weak capacity of this cell type to clear HBV infection. (Hepatology 2016;64:746-759).

Induction of atherosclerosis in mice and hamsters without germline genetic engineering.

RATIONALE: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation. OBJECTIVE: To develop a method for induction of atherosclerosis without germline genetic engineering. METHODS AND RESULTS: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters. CONCLUSIONS: Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models.

Potent microRNA suppression by RNA Pol II-transcribed 'Tough Decoy' inhibitors.

MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirus-derived gene vectors. Superior activity of two decoy-type inhibitors, a "Bulged Sponge" with eight miRNA recognition sites and a hairpin-shaped "Tough Decoy" containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.