Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation

The human DEAD-box RNA helicase DDX3 has been implicated to play a role in the whole repertoire of processes regulating gene expression, including transcription, splicing, mRNA export and translation. It has also been suggested to be involved in cell cycle control and the regulation of apoptosis. In addition, DDX3 was recently shown to be part of innate immune signalling pathways and to contribute to the induction of anti-viral mediators, such as type I interferon. Interestingly, DDX3 appears to be a prime target for viral manipulation: at least four different viruses, namely Hepatitis C virus (HCV), Hepatitis B virus (HBV), Human Immunodeficiency Virus (HIV) and poxviruses, encode proteins that interact with DDX3 andmodulate its function. HIV and HCV seem to co-opt DDX3 and require it for their replication. It has therefore been suggested that DDX3 could be a novel target for the development of drugs against these two viruses, both of which still pose major global health threats. However, in the light of the apparent multifunctionality of DDX3 in the cell, drug development strategies targeting DDX3 will have to be carefully evaluated. This review summarises the available data on the cellular functions of DDX3 and discusses theirmanipulation by the different viruses known to target DDX3. Understanding the viral strategies for manipulating or co-opting DDX3 in functional and molecular detail can provide valuable insights for the development of strategies to therapeutically target DDX3

Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKε-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through and TBK1 and consequently IFN-β induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-β induction by preventing TBK1/IKKε-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKε, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKε dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKε. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKε-mediated IRF activation and Ifnb promoter induction

DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to coordinate assembly of signalling complexes downstream of MAVS

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon induction downstream of anti-viral pattern recognition receptors (PRRs). It binds to TANK-binding kinase 1 (TBK1) and IB-kinase- (IKK, the two key kinases mediating activation of Interferon regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKK activation downstream of RIG-I and then links the activated kinase to IRF3. In this study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TRAF3 mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai Virus infection (SeV), both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the MAVS adaptor, DDX3, IKK, TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented formation of TRAF3-MAVS and TRAF3-IKKcomplexes. Based on our data, we propose that early TRAF3 ubiquitination is required for formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that coordinates assembly of different TRAF3, IKK and IRF3-containing signalling complexes downstream of MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling

DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to coordinate assembly of signalling complexes downstream of MAVS

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon induction downstream of anti-viral pattern recognition receptors (PRRs). It binds to TANK-binding kinase 1 (TBK1) and IB-kinase- (IKK, the two key kinases mediating activation of Interferon regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKK activation downstream of RIG-I and then links the activated kinase to IRF3. In this study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TRAF3 mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai Virus infection (SeV), both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the MAVS adaptor, DDX3, IKK, TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented formation of TRAF3-MAVS and TRAF3-IKKcomplexes. Based on our data, we propose that early TRAF3 ubiquitination is required for formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that coordinates assembly of different TRAF3, IKK and IRF3-containing signalling complexes downstream of MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling

A Novel IRAK1–IKKԑ Signaling Axis Limits the Activation of TAK1–IKKb Downstream of TLR3

IRAK1 is involved in the regulation of type I IFN production downstream of TLR3. Previous work indicated that IRAK1 negatively regulates TRIF-mediated activation of IRF3 and IRF7. We report that IRAK1 limits the activation of the TLR3–NF-кB pathway. Following TLR3 stimulation, IRAK1-deficient macrophages produced increased levels of IL-6 and IFN-β compared with wild type macrophages. Pharmacological inhibition of TAK1 reduced this increase in IFN-β, together with the heightened activation of IRF3 and p65 found in TLR3-ligand stimulated IRAK1-deficient macrophages. Recently, IKKԑ and TANK-binding kinase 1 (TBK1) were reported to limit activation of the NF-kB pathway downstream of IL-1R, TNFR1, and TLRs. We show that TBK1 has a positive role in the TLR3–NF-кB pathway, because we detected reduced levels of IL-6 and reduced activation of p65 in TBK1-deficient macrophages. In contrast, we show that IKK limits the activation of the TLR3–NF-кB pathway. Furthermore, we show that IRAK1 is required for the activation of IKK« downstream of TLR3. We report impaired activation of ERK1/2 in IRAK1– and IKKԑ-deficient macrophages, a novel finding for both kinases. Importantly, this work provides novel mechanistic insight into the regulation of the TLR3-signaling pathway, providing strong evidence that an IRAK1-IKKԑ–signaling axis acts to limit the production of both type I IFNs and proinflammatory cytokines by regulating TAK1 activity

TLR3 in antiviral immunity: key player or bystander?

Toll-like receptor 3 (TLR3), which recognizes double-stranded (ds)RNA, was the first identified antiviral TLR and, because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. However, this role has recently been questioned by in vivo studies and the discovery of several other antiviral pattern-recognition receptors. In this review, the function of TLR3 in the context of these other receptors, namely TLR7, 8 and 9 and the newly identified dsRNA-receptor retinoic-acid inducible gene-I (RIG-I) is discussed. Also, recent research concerning the expression profile of TLR3, its evasion by viruses and a potential role in crosspriming is addressed, which reveals a clearer appreciation of the contribution of TLR3 to antiviral immunity

Crack path selection at the interface of wrought and wire+arc additive manufactured Ti–6Al–4V

Crack propagation deviation tendency in specimens containing an interface between wrought alloy substrate and Wire + Arc Additive Manufacture (WAAM) built Ti–6Al–4V is investigated from the viewpoints of microstructure, residual stress and bi-material system. It is found that a crack initiated at the interface tends to grow into the substrate that has equiaxed microstructure and lower resistance to fatigue crack propagation. Experimental observations are interpreted by finite element modelling of the effects of residual stress and mechanical property mismatch between the WAAM and wrought alloy. Residual stresses retained in the compact tension specimens are evaluated based on measured residual stress in the initial WAAM built wall. Cracks perpendicular to the interface kept a straight path owing to the symmetrical residual stress distribution. In this case the tangential stress in bi-material model is also symmetric and has the maximum value at the initial crack plane. In contrast, cracks parallel to the interface are inclined to grow towards the substrate due to the mode II (or sliding mode) stress intensity factor caused by the asymmetric residual stress field. Asymmetric tangential stress in the bi-material model also contributes to the observed crack deviation trend according to the maximum tangential stress criterion

Squaramide—Naphthalimide Conjugates as “Turn-On” Fluorescent Sensors for Bromide Through an Aggregation-Disaggregation Approach

The syntheses of two new squaramide-naphthalimide conjugates (SQ1 and SQ2) are reported where both compounds have been shown to act as selective fluorescence “turn on” probes for bromide in aqueous DMSO solution through a disaggregation induced response. SQ1 and SQ2 displayed a large degree of self-aggregation in aqueous solution that is disrupted at increased temperature as studied by 1H NMR and Scanning Electron Microscopy (SEM). Moreover, the fluorescence behavior of both receptors was shown to be highly dependent upon the aggregation state and increasing temperature gave rise to a significant increase in fluorescence intensity. Moreover, this disaggregation induced emission (DIE) response was exploited for the selective recognition of certain halides, where the receptors gave rise to distinct responses related to the interaction of the various halide anions with the receptors. Addition of F− rendered both compounds non-emissive; thought to be due to a deprotonation event while, surprisingly, Br− resulted in a dramatic 500–600% fluorescence enhancement thought to be due to a disruption of compound aggregation and allowing the monomeric receptors to dominate in solution. Furthermore, optical sensing parameters such as limits of detection and binding constant of probes were also measured toward the various halides (F−, Cl−, Br−, and I−) where both SQ1 and SQ2 were found to sense halides with adequate sensitivity to measure μM levels of halide contamination. Finally, initial studies in a human cell line were also conducted where it was observed that both compounds are capable of being taken up by HeLa cells, exhibiting intracellular fluorescence as measured by both confocal microscopy and flow cytometry. Finally, using flow cytometry we were also able to show that cells treated with NaBr exhibited a demonstrable spectroscopic response when treated with either SQ1 or SQ2

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer