Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

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-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta 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

TRAM Is Required for TLR2 Endosomal Signaling to Type I IFN Induction

Abstract Detection of microbes by TLRs on the plasma membrane leads to the induction of proinflammatory cytokines such as TNF-α, via activation of NF-κB. Alternatively, activation of endosomal TLRs leads to the induction of type I IFNs via IFN regulatory factors (IRFs). TLR4 signaling from the plasma membrane to NF-κB via the Toll/IL-1R (TIR) adaptor protein MyD88 requires the TIR sorting adaptor Mal, whereas endosomal TLR4 signaling to IRF3 via the TIR domain–containing adaptor-inducing IFN-β (TRIF) requires the TRIF-related adaptor molecule (TRAM). Similar to TLR4 homodimers, TLR2 heterodimers can also induce both proinflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, whereas endosomal IRF activation by TLR2 requires MyD88. However, it was unclear whether TLR2 requires a sorting adaptor for endosomal signaling, like TLR4 does. In this study, we show that TLR2-dependent IRF7 activation at the endosome is both Mal- and TRAM-dependent, and that TRAM is required for the TLR2-dependent movement of MyD88 to endosomes following ligand engagement. TRAM interacted with both TLR2 and MyD88, suggesting that TRAM can act as a bridging adapter between these two molecules. Furthermore, infection of macrophages lacking TRAM with herpes viruses or the bacterium Staphylococcus aureus led to impaired induction of type I IFN, indicating a role for TRAM in TLR2-dependent responses to human pathogens. Our work reveals that TRAM acts as a sorting adaptor not only for TLR4, but also for TLR2, to facilitate signaling to IRF7 at the endosome, which explains how TLR2 is capable of causing type I IFN induction.</jats:p

Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways

Studies into the effects of the vaccinia virus protein A46R on Interleukin-1/Toll-Like receptor signalling pathways

THESIS 7692Poxviruses circumvent the host immune response by encoding proteins that can antagonise host defence mechanisms. The vaccinia virus (VV) protein, A46R, was identified based on sequence similarity to the Toll/Interleukin-1/Resistance homology (TIR) domain, the cytosolic signalling domain of the interleukin-1 receptor (IL-IR ) and Toll-like receptors (TLRs). TLRs play a crucial role in host defence against invading micro-organisms by mediating pro-inflammatory signalling cascades, mainly through their cytoplasmic TIR domains. A46R is the only viral protein so far identified to have a TIR domain. Initial studies revealed that A46R could inhibit IL-1, but not tumour necrosis factor (TNF) a-induced NFkB activation (Bowie et al, 2000), suggesting a role for the protein in immune evasion. This study shows that A46R also blocked multiple signals em anating from TLR4, suggesting that it was acting on a molecule close to the receptor complex that was also required for IL-1 signalling. Immunoprecipitation and GST-pulldown experiments revealed that A46R targeted MyD88, a TIR adaptor molecule used by both IL-1 and TLR4, while further studies showed A46R blocked MyD88-dependent pathways in murine macrophages. This is the first example of a viral protein targeting MyD88

The human adaptor SARM negatively regulates adaptor protein TRIF–dependent Toll-like receptor signaling

Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll–interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction ‘downstream’ of TRIF but not of MyD88. SARM associated with TRIF, and ‘knockdown’ of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling

Peptide identification from a<i>Porphyra dioica</i>protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities

Novel bioactive peptides were identified from aPorphyra dioicaprotein hydrolysate.</p