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Climate warming is altering snowpack permanence in alpine tundra, modifying shrub growth and distribution. Plant acclimation to snowpack changes depends on the capability to guarantee growth and carbon storage, suggesting that the content of non-structural carbohydrates (NSC) in plant organs can be a key trait to depict the plant response under different snow regimes. To test this hypothesis, we designed a 3-years long manipulative experiment aimed at evaluating the effect of snow melt timing (i.e., early, control, and late) on NSC content in needles, bark and wood of Juniperus communis L. growing at high elevation in the Alps. Starch evidenced a general decrease from late spring to summer in control and early melting, while starch was low but stable in plants subjected to a late snow melt. Leaves, bark and wood have different level of soluble NSC changing during growing season: in bark, sugars content decreased significantly in late summer, while there was no seasonal effect in needles and wood. Soluble NSC and starch were differently related with the plant growth, when considering different tissues and snow treatment. In leaf and bark we observed a starch depletion in control and early melting plants, consistently to a higher growth (i.e., twig elongation), while in late snow melt, we did not find any significant relationship between growth and NSC concentration. Our findings confirmed that snowpack duration affects the onset of the growing season promoting a change in carbon allocation in plant organs and, between bark and wood in twigs. Finally, our results suggest that plants, at this elevation, could take advantage from an early snow melt caused by climate warming, most likely due to photosynthetic activity by maintaining the level of reserves and enhancing the carbon investment for growth., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Therapies that modulate and appropriately direct the immune response are promising candidates for the treatment of infectious diseases. One such candidate therapeutic is DZ13, a short, synthetic, single-stranded DNA molecule. This molecule has enzymatic activity and can modulate the immune response by binding to and degrading the mRNA encoding a key immuno-regulatory molecule. Originally developed and entering clinical trials as an anti-cancer agent, DZ13 has also been evaluated as a treatment for viral infections, and has been shown to provide protection against infection with influenza virus in a mouse model of infection. In this work, we evaluated whether the immuno-modulatory properties of DZ13 could provide protection against the potential biothreat pathogen Burkholderia pseudomallei which causes the neglected tropical disease melioidosis. Treatment of mice infected with B. pseudomallei demonstrated that DZ13 did indeed provide excellent protection after only two post-exposure treatments. However, our data indicated that the enzymatic activity contained in DZ13 was not required for protection, with control oligonucleotide treatments lacking activity against the target mRNA equally as protective against B. pseudomallei . We have designed new sequences to study the mechanism of protection further. These novel sequences offer enhanced protection against infection, but are not directly anti-microbial and do not appear to be stimulating the immune system via TLR9 or other key innate immune sensors, despite containing CpG motifs. The molecular mechanism of these novel sequences remains to be elucidated, but the data highlights that these oligonucleotide-sensing pathways are attractive and relevant targets to modulate during bacterial and viral infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Scott, Farrar, Young, Prior, Stratilo, Unterholzner and D’Elia.)

Climate change impacts on forest trees will be particularly severe for relict species endemic to the subalpine forest, such as Pinus cembra in the Alps and Carpathians. Most current knowledge about the response of this species to climate comes from tree-ring width analysis. However, this approach cannot perform in-depth and highly time-resolved analysis on the climate influence on specific growth processes and xylem functions. We analyzed xylem anatomical traits from six sites covering most of the longitudinal range of this species. Associations between climate and cell number, lumen area and cell wall thickness were computed for the 1920-2010 period using climate records aligned to degree-day temperature sum thresholds. The anatomical chronologies were clearly distinct between the Alps and Carpathians. However, climate responses were similar for all sites, suggesting common species-specific response mechanisms. Temperature showed a positive correlation with both cell number and cell wall thickness. Cell lumen size exhibited an early positive association, followed by strong negative association with temperature and a positive one with precipitation. This highlights that the cell enlargement process was negatively related to high temperature at high elevation, where meristematic processes are rather supposed to be constrained by low temperatures. Therefore, long-term climate warming can have negative consequences on the xylem potential to transport water at all investigated sites. Moreover, in the last 30 years, we observed a slight anticipation of some responses and a decrease in climate sensitivity of some xylem parameters. Our findings provide evidence of temporally unstable but spatially consistent climate response of Pinus cembra from the Alps to the Carpathians. The low diversity in xylem phenotypic responses to climate suggests that future warming could extensively and evenly affect the species throughout its entire distribution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Forests are threatened by increasingly severe and more frequent drought events worldwide. Mono-specific forests, developed as a consequence of widespread management practices established early last century, seem particularly susceptible to global warming and drought compared with mixed-species forests. Although, in several contexts, mixed-species forests display higher species diversity, higher productivity, and higher resilience, previous studies highlighted contrasting findings, with not only many positive but also neutral or negative effects on tree performance that could be related to tree species diversity. Processes underlying this relationship need to be investigated. Wood anatomical traits are informative proxies of tree functioning, and they can potentially provide novel long-term insights in this regard. However, wood anatomical traits are critically understudied in such a context. Here, we assess the role of tree admixture on Pinus sylvestris L. xylem traits such as mean hydraulic diameter, cell wall thickness, and anatomical wood density, and we test the variability of these traits in response to climatic parameters such as temperature, precipitation, and drought event frequency and intensity., Methods: Three monocultural plots of P. sylvestris and three mixed-stand plots of P. sylvestris and Quercus sp. were identified in Poland and Spain, representing Continental and Mediterranean climate types, respectively. In each plot, we analyzed xylem traits from three P. sylvestris trees, for a total of nine trees in monocultures and nine in mixed stands per study location., Results: The results highlighted that anatomical wood density was one of the most sensitive traits to detect tree responses to climatic conditions and drought under different climate and forest types. Inter-specific facilitation mechanisms were detected in the admixture between P. sylvestris and Quercus sp., especially during the early growing season and during stressful events such as spring droughts, although they had negligible effects in the late growing season., Discussion: Our findings suggest that the admixture between P. sylvestris and Quercus sp. increases the resilience of P. sylvestris to extreme droughts. In a global warming scenario, this admixture could represent a useful adaptive management option., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Giberti, von Arx, Giovannelli, du Toit, Unterholzner, Bielak, Carrer, Uhl, Bravo, Tonon and Wellstein.)

Current global change is inducing heterogeneous warming trends worldwide, with faster rates at higher latitudes in the Northern Hemisphere. Consequently, tundra vegetation is experiencing an increase in growth rate and uneven but expanding distribution. Yet, the drivers of this heterogeneity in woody species responses are still unclear. Here, applying a retrospective approach and focusing on long-term responses, we aim to get insight into growth trends and climate sensitivity of long-lived woody species belonging to different functional types with contrasting growth forms and leaf habits (shrub vs. tree and deciduous vs. evergreen). A total of 530 samples from 7 species (common juniper, dwarf birch, woolly willow, Norway spruce, lodgepole pine, rowan, and downy birch) were collected in 10 sites across Iceland. We modelled growth trends and contrasted yearly ring-width measurements, filtering in high- and low-frequency components, with precipitation, land- and sea-surface temperature records (1967-2018). Shrubs and trees showed divergent growth trends, with shrubs closely tracking the recent warming, whereas trees, especially broadleaved, showed strong fluctuations but no long-term growth trends. Secondary growth, particularly the high-frequency component, was positively correlated with summer temperatures for most of the species. On the contrary, growth responses to sea surface temperature, especially in the low frequency, were highly diverging between growth forms, with a strong positive association for shrubs and a negative for trees. Within comparable vegetation assemblage, long-lived woody species could show contrasting responses to similar climatic conditions. Given the predominant role of oceanic masses in shaping climate patterns in the Arctic and Low Arctic, further investigations are needed to deepen the knowledge on the complex interplay between coastal tundra ecosystems and land-sea surface temperature dynamics., (© 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

The DNA sensor cGAS (cyclic GMP-AMP synthase) and its adaptor protein STING (Stimulator of Interferon Genes) detect the presence of cytosolic DNA as a sign of infection or damage. In cancer cells, this pathway can be activated through persistent DNA damage and chromosomal instability, which results in the formation of micronuclei and the exposure of DNA fragments to the cytosol. DNA damage from radio- or chemotherapy can further activate DNA sensing responses, which may occur in the cancer cells themselves or in stromal and immune cells in the tumour microenvironment (TME). cGAS-STING signalling results in the production of type I interferons, which have been linked to immune cell infiltration in 'hot' tumours that are susceptible to immunosurveillance and immunotherapy approaches. However, recent research has highlighted the complex nature of STING signalling, with tumours having developed mechanisms to evade and hijack this signalling pathway for their own benefit. In this mini-review we will explore how cGAS-STING signalling in different cells in the TME can promote both anti-tumour and pro-tumour responses. This includes the role of type I interferons and the second messenger cGAMP in the TME, and the influence of STING signalling on local immune cell populations. We examine how alternative signalling cascades downstream of STING can promote chronic interferon signalling, the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the production of inflammatory cytokines, which can have pro-tumour functions. An in-depth understanding of DNA sensing in different cell contexts will be required to harness the anti-tumour functions of STING signalling., (© 2023 The Author(s).)

The DNA sensor cGAS and its adaptor STING constitute an ancient pathogen detection mechanism, but it is unclear to what extent its function is conserved across the animal kingdom. In this issue of Cell, Kranzusch and colleagues identify thousands of cGAS-like receptors and discover networks of second messengers that activate innate immune responses in animals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

High-elevation ecosystems are one of the most sensitive to climate change. The analysis of growth and xylem structure of trees from marginal populations, especially the ones growing at the treeline, could provide early-warning signs to better understand species-specific responses to future climate conditions. In this study, we combined classical dendrochronology with wood density and anatomical measurements to investigate the climate sensitivity of Pinus cembra L., a typical European high-elevation tree species distributed in isolated patches in the Carpathians. Samples were collected from the Retezat Mountains, South-Western Romania. We analyzed ring width (TRW), maximum density (MXD), xylem anatomical traits [cell number per ring (CNo), cell density (CD), conduit area (CA), and cell wall thickness (CWT)] time series, split into ring sectors and assessed the relationships with monthly and daily climate records over the last century (1901-2015). The analysis showed a strong dependency of TRW on CNo and MXD on CWT. Summer temperature positively correlated with MXD and CWT [monthly correlation ( r) were 0.65 and 0.48 respectively] from the early to late wood but not TRW ( r = 0.22). CA positively correlated with water availability ( r = 0.37) and negatively correlated with temperature ( r = -0.39). This study improves our general understanding of the climate-growth relationships of a European high-elevation tree species and the results could be considered for forecasting population dynamics on projected changes in climate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Știrbu, Roibu, Carrer, Mursa, Unterholzner and Prendin.)

With the recent climate warming, tundra ecotones are facing a progressive acceleration of spring snowpack melting and extension of the growing season, with evident consequences to vegetation. Along with summer temperature, winter precipitation has been recently recognised as a crucial factor for tundra shrub growth and physiology. However, gaps of knowledge still exist on long-living plant responses to different snowpack duration, especially on how intra-specific and year-to-year variability together with multiple functional trait adjustments could influence the long-term responses. To fill this gap, we conducted a 3 years snow manipulation experiment above the Alpine treeline on the typical tundra species Juniperus communis , the conifer with the widest distributional range in the north emisphere. We tested shoot elongation, leaf area, stomatal density, leaf dry weight and leaf non-structural carbohydrate content of plants subjected to anticipated, natural and postponed snowpack duration. Anticipated snowpack melting enhanced new shoot elongation and increased stomatal density. However, plants under prolonged snow cover seemed to compensate for the shorter growing period, likely increasing carbon allocation to growth. In fact, these latter showed larger needles and low starch content at the beginning of the growing season. Variability between treatments slightly decreased over time, suggesting a progressive acclimation of juniper to new conditions. In the context of future warming scenarios, our results support the hypothesis of shrub biomass increase within the tundra biome. Yet, the picture is still far from being complete and further research should focus on transient and fading effects of changing conditions in the long term., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Unterholzner, Prendin, Dibona, Menardi, Casolo, Gargiulo, Boscutti and Carrer.)

Type I interferons (IFNs) are critical for anti-viral responses, and also drive autoimmunity when dysregulated. Upon viral sensing, monocytes elicit a sequential cascade of IFNβ and IFNα production involving feedback amplification, but how exactly this cascade is regulated in human cells is incompletely understood. Here we show that the PYHIN protein myeloid cell nuclear differentiation antigen (MNDA) is required for IFNα induction in monocytes. Unlike other PYHINs, this is not due to a pathogen sensing role, but rather MNDA regulated expression of IRF7, a transcription factor essential for IFNα induction. Mechanistically, MNDA is required for recruitment of STAT2 and RNA polymerase II to the IRF7 gene promoter, and in fact MNDA is itself recruited to the IRF7 promoter after type I IFN stimulation. These data implicate MNDA as a critical regulator of the type I IFN cascade in human myeloid cells and reveal a new role for human PYHINs in innate immune gene induction., (© 2022. The Author(s).)

During winter, conifers at the alpine treeline suffer dramatic losses of hydraulic conductivity, which are successfully recovered during late winter. Previous studies indicated branch water uptake to support hydraulic recovery. We analyzed water absorption and redistribution in Picea abies and Larix decidua growing at the treeline by in situ exposure of branches to δ 2 H-labelled water. Both species suffered high winter embolism rates (> 40-60% loss of conductivity) and recovered in late winter (< 20%). Isotopic analysis showed water to be absorbed over branches and redistributed within the crown during late winter. Labelled water was redistributed over 425 ± 5 cm within the axes system and shifted to the trunk, lower and higher branches (tree height 330 ± 40 cm). This demonstrated relevant branch water uptake and re-distribution in treeline conifers. The extent of water absorption and re-distribution was species-specific, with L. decidua showing higher rates. In natura, melting snow might be the prime source for absorbed and redistributed water, enabling embolism repair and restoration of water reservoirs prior to the vegetation period. Pronounced water uptake in the deciduous L. decidua indicated bark to participate in the process of water absorption., (© 2021. The Author(s).)

Rabies represents a typical model for spillover of zoonotic viral diseases among multiple hosts. Understanding the success of rabies virus (RV) in switching hosts requires the analysis of viral evolution and host interactions. In this study, we have investigated the structural and sequence analysis of host receptors among different RV susceptible host species. Our extensive bioinformatic analysis revealed the absence of the integrin plexin domain in the integrin β1 (ITGB1) receptor of the black fruit bats in the current annotation of the genome. Interestingly, the nicotinic acetyl choline receptor (nAChR) interaction site with the glycoprotein (G) of RV was conserved among different species. To study the interaction dynamics between RV-G protein and the RV receptors, we constructed and analyzed structures of RV receptors and G proteins using homology modeling. The molecular docking of protein-protein interaction between RV-G protein and different host receptors highlighted the variability of interacting residues between RV receptors of different species. These in silico structural analysis and interaction mapping of viral protein and host receptors establish the foundation to understand complex entry mechanisms of RV entry, which may facilitate the understanding of receptor mediated spillover events in RV infections and guide the development of novel vaccines to contain the infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Khalifa, Unterholzner and Munir.)

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The performance and distribution of woody species strongly depend on their adjustment to environmental conditions based on genotypic and phenotypic properties. Since more intense and frequent drought events are expected due to climate change, xylem hydraulic traits will play a key role under future conditions, and thus, knowledge of hydraulic variability is of key importance. In this study, we aimed to investigate the variability in hydraulic safety and efficiency of the conifer shrub Juniperus communis based on analyses along an elevational transect and a common garden approach. We studied (i) juniper plants growing between 700 and 2000 m a.s.l. Innsbruck, Austria, and (ii) plants grown in the Innsbruck botanical garden (Austria) from seeds collected at different sites across Europe (France, Austria, Ireland, Germany and Sweden). Due to contrasting environmental conditions at different elevation and provenance sites and the wide geographical study area, pronounced variation in xylem hydraulics was expected. Vulnerability to drought-induced embolisms (hydraulic safety) was assessed via the Cavitron and ultrasonic acoustic emission techniques, and the specific hydraulic conductivity (hydraulic efficiency) via flow measurements. Contrary to our hypothesis, relevant variability in hydraulic safety and efficiency was neither observed across elevations, indicating a low phenotypic variation, nor between provenances, despite expected genotypic differences. Interestingly, the provenance from the most humid and warmest site (Ireland) and the northernmost provenance (Sweden) showed the highest and the lowest embolism resistance, respectively. The hydraulic conductivity was correlated with plant height, which indicates that observed variation in hydraulic traits was mainly related to morphological differences between plants. We encourage future studies to underlie anatomical traits and the role of hydraulics for the broad ecological amplitude of J. communis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Self-DNA has previously been thought to be protected from immune detection by compartmentalisation in the nucleus or mitochondria. Here, we describe the discovery of a signalling cascade that links the detection of DNA damage in the nucleus to the activation of the innate immune adaptor STING (STimulator of INterfern Genes).

Intracellular DNA and RNA sensors play a vital part in the innate immune response to viruses and other intracellular pathogens, causing the secretion of type I interferons, cytokines and chemokines from infected cells. Pathogen RNA can be detected by retinoic-acid inducible gene I-like receptors in the cytosol, whereas cytosolic DNA is recognized by DNA sensors such as cyclic GMP-AMP synthase (cGAS). The resulting local immune response, which is initiated within hours of infection, is able to eliminate many pathogens before they are able to establish an infection in the host. For this reason, all viruses, and some intracellular bacteria and protozoa, need to evade detection by nucleic acid sensors. Immune evasion strategies include the sequestration and modification of nucleic acids, and the inhibition or degradation of host factors involved in innate immune signalling. Large DNA viruses, such as herpesviruses, often use multiple viral proteins to inhibit signalling cascades at several different points; for instance herpes simplex virus 1 targets both DNA sensors cGAS and interferon-γ-inducible protein 16, as well as the adaptor protein STING (stimulator of interferon genes) and other signalling factors in the pathway. Viruses with a small genome encode only a few immunomodulatory proteins, but these are often multifunctional, such as the NS1 protein from influenza A virus, which inhibits RNA sensing in multiple ways. Intracellular bacteria and protozoa can also be detected by nucleic acid sensors. However, as the type I interferon response is not always beneficial for the host under these circumstances, some bacteria subvert, rather than evade, these signalling cascades for their own gain., (© 2018 The Authors. Immunology Published by John Wiley & Sons Ltd.)

DNA damage can be sensed as a danger-associated molecular pattern by the innate immune system. Here we find that keratinocytes and other human cells mount an innate immune response within hours of etoposide-induced DNA damage, which involves the DNA sensing adaptor STING but is independent of the cytosolic DNA receptor cGAS. This non-canonical activation of STING is mediated by the DNA binding protein IFI16, together with the DNA damage response factors ATM and PARP-1, resulting in the assembly of an alternative STING signaling complex that includes the tumor suppressor p53 and the E3 ubiquitin ligase TRAF6. TRAF6 catalyzes the formation of K63-linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. We propose that STING acts as a signaling hub that coordinates a transcriptional response depending on its mode of activation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Climate sensitivity of populations at the margins of their distribution range is of key importance to understand species' responses to future warming conditions. Pinus cembra is of particular interest being a typical high-elevation taxon, spread with mostly scattered populations within its actual range, but still overlooked in traditional dendrochronological researches due to low tree-ring variability and climate sensitivity. With a different approach, we analyzed time series of xylem anatomical traits, split into intra-ring sectors, and used daily climate records over 89 years (1926-2014) aiming to improve the quality and time resolution of the climate/growth associations. From nine trees growing at their altitudinal limit and on 1.5 × 10 6 tracheids, we measured ring width (MRW), cell number per ring, lumen area (LA), and cell-wall thickness (CWT). We then computed correlations with monthly and fortnightly climate data. Late-spring and summer temperature emerged as the most important factors. LA and especially CWT showed a stronger temperature response than MRW, starting in mid-May and early June, respectively. CWT also evidenced the longest period of correlations with temperature and a significant difference between latewood radial and tangential walls. Analysis of xylem anatomical traits at intra-ring level and the use of daily temperature records proved to be useful for high resolution and detailed climate/growth association inferences in Pinus cembra.

The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-γ-inducible 16 is important for induction of IFN-β in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-γ-inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

It has been assumed that cells distinguish viral from cellular DNA due to the former's presence in the cytosol. However, in this issue, Kerur et al. (2011) propose that the DNA genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is recognized inside the nucleus by the DNA sensor IFI16, leading inflammasome activation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly associated with IFN-β-inducing viral DNA motifs. STING, a critical mediator of IFN-β responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-β induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).

The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.

The immediate response to viral infection relies on pattern-recognition receptors (PRRs), most prominently the Toll-like receptors (TLRs) and the RNA helicases RIG-I and MDA5, as well as double stranded RNA-dependent protein kinase (PKR) and the DNA receptor, DAI. These PRRs recognize pathogen-associated molecular patterns (PAMPs) such as viral proteins and nucleic acids. The engagement of these receptors then initiates intracellular signaling cascades which ultimately cause the activation of transcription factors and the expression of type I interferons and pro-inflammatory cytokines. This innate response establishes an anti-viral state in the infected cell and its neighbours and alerts immune cells to the danger. In order to establish a productive infection, viruses need to overcome this initial anti-viral response. Evasion of innate immune defences is achieved by means of viral proteins that inhibit the signaling cascades emanating from the PRRs. The same innate signal transduction pathways have been implicated in conditions of sterile inflammation, such as rheumatoid arthritis and multiple sclerosis, and in autoimmunity. Because viral proteins target crucial host proteins involved in these pathways, they can point the way to key drug targets. Further, the viral proteins themselves or derivatives of them may be of use therapeutically to curtail inflammation and autoimmunity.

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that eliminates mRNAs containing premature termination codons (PTCs). The proteins UPF1, SMG5, SMG6, and SMG7 are essential NMD factors in metazoa. SMG5 and SMG7 form a complex with UPF1 and interact with each other via their N-terminal domains. Here we show that SMG5 and SMG7 colocalize in cytoplasmic mRNA decay bodies, while SMG6 forms separate cytoplasmic foci. When SMG7 is tethered to a reporter transcript, it elicits its degradation, bypassing the requirement for a PTC, UPF1, SMG5, or SMG6. This activity is mediated by the C-terminal domain of SMG7. In contrast, SMG5 requires SMG7 to trigger mRNA decay and to localize to decay bodies. Our findings indicate that SMG7 provides a link between the NMD and the mRNA degradation machinery by interacting with SMG5 and UPF1 via its N-terminal domain and targeting bound transcripts for decay via its C-terminal domain.

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago-Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago-Y14. Here we show that PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago-Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 A resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD.

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified. Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in Drosophila. Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.