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 IKKe and TBK1 and consequently IFN-b 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-b induction by preventing TBK1/IKKe-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/IKKe, 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/IKKe 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 IKKe. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKe-mediated IRF activation and Ifnb promoter induction.

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Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like–interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain–containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain–containing adaptor inducing IFN-β (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor κB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-β by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

a b s t r a c t a r t i c l e i n f oTraditional functions of DExD/H-box helicases are concerned with RNA metabolism; they have been shown to play a part in nearly every cellular process that involves RNA. On the other hand, it is accepted that DexD/H-box helicases also engage in activities that do not require helicase activity. A number of DExD/ H-box helicases have been shown to be involved in anti-viral immunity. The RIG-like helicases, RIG-I, mda5 and lgp2, act as important cytosolic pattern recognition receptors for viral RNA. Detection of viral nucleic acids by the RIG-like helicases or other anti-viral pattern recognition receptors leads to the induction of type I interferons and pro-inflammatory cytokines. More recently, additional DExD/H-box helicases have also been implicated to act as cytosolic sensors of viral nucleic acids, including DDX3, DDX41, DHX9, DDX60, DDX1 and DHX36. However, there is evidence that at least some of these helicases might have more downstream functions in pattern recognition receptor signalling pathways, as signalling adaptors or transcriptional regulators. In an interesting twist, a lot of DExD/H-box helicases have also been identified as essential host factors for the replication of different viruses, suggesting that viruses 'hijack' their RNA helicase activities for their benefit. Interestingly, DDX3, DDX1 and DHX9 are among the helicases that are required for the replication of a diverse range of viruses. This might suggest that these helicases are highly contested targets in the ongoing 'arms race' between viruses and the host immune system. This article is part of a Special Issue entitled: The Biology of RNA helicases -Modulation for life.

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.

To cite this version:Martina Schröder. Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation. Biochemical Pharmacology, Elsevier, 2009, 79 (3) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 42A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 AbstractThe 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 and modulate 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 their manipulation 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.

The human DEAD box protein 3 (DDX3) has been implicated in different processes contributing to gene expression. Interestingly, DDX3 is required as an essential host factor for the replication of HIV and hepatitis C virus (HCV) and is therefore considered a potential drug target. On the other hand, DDX3 interacts with IB kinase (IKK) and TANK-binding kinase 1 (TBK1) and contributes to the induction of antiviral type I interferons (IFNs). However, the molecular mechanism by which DDX3 contributes to IFN induction remains unclear. Here we show that DDX3 mediates phosphorylation of interferon regulatory factor 3 (IRF3) by the kinase IKK. DDX3 directly interacts with IKK and enhances its autophosphorylation and activation. IKK then phosphorylates several serine residues in the N terminus of DDX3. Phosphorylation of DDX3 at serine 102 (S102) is required for recruitment of IRF3 to DDX3, facilitating its phosphorylation by IKK. Mutation of S102 to alanine disrupted the interaction between DDX3 and IRF3 but not that between DDX3 and IKK. The S102A mutation failed to enhance ifnb promoter activation, suggesting that the DDX3-IRF3 interaction is crucial for this effect. Our data implicates DDX3 as a scaffolding adaptor that directly facilitates phosphorylation of IRF3 by IKK. DDX3 might thus be involved in pathway-specific activation of IRF3. The human DEAD box RNA helicase DDX3 is a multifunctional cellular protein which has been implicated in various processes linked to gene expression (1). Its RNA helicase activity is coopted by viruses that require DDX3 as an essential host factor for their replication, such as HIV and HCV (2-4). In contrast, we and others have previously demonstrated that DDX3 contributes to antiviral innate immune signaling pathways leading to ifnb induction (5-9). This function of DDX3 is independent of its ATPase and helicase activity (6, 7). ifnb promoter induction requires activation of the transcription factors IRF3 and IRF7, which occurs only downstream of certain, mostly antiviral, pattern recognition receptors (PRRs). Antiviral PRRs comprise the endosomal Toll-like receptors TLR3, TLR7, TLR8, and TLR9, cytosolic RIG-like helicases (RLHs), and several newly discovered cytosolic DNA receptors (10). These receptors recognize different species of viral nucleic acids and induce type I IFNs in response (11). TLR3 and TLR4 engage the TRIF (TIR domain-containing adaptor inducing IFN-␤)-dependent pathway for activation of IRF3 (12). In this pathway, phosphorylation-dependent activation of IRF3 is mediated by the IKK-related kinases TBK1 and IKKε (13,14). The RLHs also utilize TBK1 and IKKε for IRF3 activation after engaging the adaptor molecule MAVS (mitochondrial antiviral signaling) (15, 16). DNA receptors activate IRF3 through TBK1 and the adaptor molecule STING (stimulator of IFN genes).While these signaling pathways converge on IKKε and TBK1 for IRF3 activation, not all receptors that activate IKKε and/or TBK1 lead to IRF3 activation (17). This suggests that additional factors are required for li...

Poxviruses have evolved numerous strategies to evade host innate immunity. Vaccinia virus K7 is a 149-residue protein with previously unknown structure that is highly conserved in the orthopoxvirus family. K7 bears sequence and functional similarities to A52, which interacts with interleukin receptor-associated kinase 2 and tumor necrosis factor receptor-associated factor 6 to suppress nuclear factor kappaB activation and to stimulate the secretion of the anti-inflammatory cytokine interleukin-10. In contrast to A52, K7 forms a complex with DEAD box RNA helicase DDX3, thereby suppressing DDX3-mediated ifnb promoter induction. We determined the NMR solution structure of K7 to provide insight into the structural basis for poxvirus antagonism of innate immune signaling. The structure reveals an alpha-helical fold belonging to the Bcl-2 family despite an unrelated primary sequence. NMR chemical-shift mapping studies have localized the binding surface for DDX3 on a negatively charged face of K7. Furthermore, thermodynamic studies have mapped the K7-binding region to a 30-residue N-terminal fragment of DDX3, ahead of the core RNA helicase domains.