Replication of lentiviruses generates different DNA forms, including RNA:DNA hybrids, ssDNA, and dsDNA. Nucleic acids stimulate innate immune responses, and pattern recognition receptors detecting ...dsDNA have been identified. However, sensors for ssDNA have not been reported, and the ability of RNA:DNA hybrids to stimulate innate immune responses is controversial. Using ssDNAs derived from HIV-1 proviral DNA, we report that this DNA form potently induces the expression of IFNs in primary human macrophages. This response was stimulated by stem regions in the DNA structure and was dependent on IFN-inducible protein 16 (IFI16), which bound immunostimulatory DNA directly and activated the stimulator of IFN genes –TANK-binding kinase 1 - IFN regulatory factors 3/7 (STING–TBK1–IRF3/7) pathway. Importantly, IFI16 colocalized and associated with lentiviral DNA in the cytoplasm in macrophages, and IFI16 knockdown in this cell type augmented lentiviral transduction and also HIV-1 replication. Thus, IFI16 is a sensor for DNA forms produced during the lentiviral replication cycle and regulates HIV-1 replication in macrophages.
IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are ...transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.
Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, ...and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.
Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral ...infection. However, it is not known how HSV‐1 inhibits IFN expression in this cell type. Here, we show that HSV‐1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV‐1 inhibits expression of type I IFN in human macrophages through ICP27‐dependent targeting of the TBK1‐activated STING signalsome.
Synopsis
Type I interferon (IFN) is important for control of infection with viruses, including herpes simplex virus (HSV)‐1. DNA viruses, such as HSV‐1, induce IFN expression after sensing of viral DNA by cGAS and signaling through the STING–TBK1–IRF3 signaling pathway. Here, we demonstrate that the HSV‐1 protein ICP27 targets the STING‐TBK1 signalsome and inhibits IRF3 activation, thus preventing IFN expression.
ICP27 prevents activation of IRF3 but not TBK1.
ICP27 is translocated to the cytoplasm at late time points of infection where is co‐localizes with TBK1 and STING.
ICP27 associates with STING and TBK1 in a manner dependent on expression of both host proteins.
ICP27 from the Simplexvirus genera of Alphaherpesvirinae inhibits IFN production in a manner dependent on the RGG box.
Herpes simplex virus evades cGAS/STING/TBK1‐mediated innate immunity activation in human macrophages by nuclear translocation of its ICP27 effector protein and suppression of IRF3 signaling.
Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by recurrent bacterial infections and defined by reduced levels of IgG, IgA, and/or IgM, insufficient response to ...polysaccharide vaccination, and an abnormal B-cell immunophenotype with a significantly reduced fraction of isotype-switched memory B cells. In addition to this infectious phenotype, at least one third of the patients experience autoimmune, autoinflammatory, granulomatous, and/or malignant complications. The very heterogeneous presentation strongly suggests a collection of different disease entities with somewhat different pathogeneses and most likely diverse genetic etiologies. Major progress has been made during recent years with the advent and introduction of next-generation sequencing, initially for research purposes, but more recently in clinical practice. In the present study, we performed whole exome sequencing on 20 CVID patients with autoimmunity, autoinflammation, and/or malignancy from the Danish CVID cohort with the aim to identify gene variants with a certain, possible, or potential disease-causing role in CVID. Through bioinformatics analyses, we identified variants with possible/probable disease-causing potential in nine of the patients. Of these, three patients had four variants in three different genes classified as likely pathogenic (
, and
), whereas in six patients, we identified seven variants of possible pathogenic potential classified as variants of unknown significance (
, and
). In the remaining 11 patients, we did not identify possible genetic causes. Genetic findings were correlated to clinical disease presentation, clinical immunological phenotype, and disease complications. We suggest that the variants identified in the present work should lay the ground for future studies to functionally validate their disease-causing potential and to investigate at the mechanistic and molecular level their precise role in CVID pathogenesis. Overall, we believe that the present work contributes important new insights into the genetic basis of CVID and particular in the subset of CVID patients with a complex phenotype involving not only infection, but also autoimmunity, autoinflammation, and malignancy.
The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic ...reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.
Since April 2014, an outbreak of influenza in harbor seals has been ongoing in northern Europe. In Denmark during June-August, 152 harbor seals on the island of Anholt were found dead from severe ...pneumonia. We detected influenza A(H10N7) virus in 2 of 4 seals examined.
Aleutian mink disease (AMD) is a chronic viral disease in farmed mink and the virus (AMDV) has been found in many free-ranging mink (Neovison vison) populations in Europe and North America. In this ...study, AMDV DNA and AMDV antibodies were analysed in 144 free-ranging mink hunted in Sweden. Associations between being AMDV infected (defined as positive for both viral DNA and antibodies) and the weight of the spleen, liver, kidneys, adrenal glands and body condition were calculated and the sequences of ten AMDV isolates were analysed in order to characterize the genetic relationships. In total, 46.1% of the mink were positive for AMDV antibodies and 57.6% were positive for AMDV DNA. Twenty-two percent of the mink tested on both tests (n = 133) had dissimilar results. The risk of having AMDV antibodies or being positive for AMDV DNA clearly increased with age and the majority of the mink that were two years or older were infected. Few macroscopic changes were found upon necropsy. However, the relative weight of the spleen was sexually dimorphic and was found to be slightly, but significantly (p = 0.006), heavier in AMDV infected male mink than uninfected. No association between AMDV infection and body condition, weight of the kidneys, liver or adrenal glands were found. Several different strains of AMDV were found across the country. Two of the AMDV sequences from the very north of Sweden did not group with any of the previously described groups of strains. In summary, AMDV seems to be prevalent in wild mink in Sweden and may subtly influence the weight of the spleen.
Background
During the COVID‐19 pandemic focus has been on polyethylene glycol (PEG) and polysorbate as these excipients are constituents in the first vaccines and possible elicitors of allergic ...reactions to the vaccines. We aimed to evaluate the possibility of vaccinating patients with PEG and/or polysorbate allergy against COVID‐19.
Methods
Twenty‐five patients with a history of an allergic reaction to drugs, vaccines and mouth hygiene products containing PEG or polysorbate and sensitization (skin test or in vitro test) or a positive challenge were included. We re‐evaluated 19 of 21 patients diagnosed before 2021 and four new patients by skin prick tests (SPT) and Basophil Histamine Release (BaHR) for PEGs, polysorbates and approved COVID‐19 vaccines as well as measurement of specific IgE (PEG 2000, 10,000). Patients were offered vaccination based on decision points from the primary diagnosis and re‐evaluation.
Results
Most common primary elicitors were depot‐steroids and laxatives. Most patients had experienced more than one reaction. SPT was superior to BaHR test although many SPTs became negative over time. After careful re‐evaluation three patients were successfully vaccinated with the Pfizer/BioNTech vaccine. Three were vaccinated before referral. Eleven were offered the Johnson‐Johnson vaccine; four were vaccinated successfully, seven abstained. Six patients could not be vaccinated with PEG or polysorbate containing vaccines.
Conclusion
Hypersensitivity to excipients in COVID‐19 vaccines constitutes a risk to patients with allergy to PEG or polysorbates. After diagnostic evaluation, a safe COVID‐19 vaccine could be offered to most patients, the remainders will await new vaccines containing different excipients.
Hepatitis E virus (HEV) is a zoonotic virus for which pigs are the primary animal reservoir. To investigate whether HEV occurs in mink in Denmark, we screened feces and tissues from domestic and wild ...mink. Our finding of a novel HEV variant supports previous findings of HEV variants in a variety of species.
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