No abstract

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.

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 domaincontaining adapter molecules transduces signals from engaged receptors that ultimately activate NF-B and/or interferon regulatory factor 3 (IRF3) to induce proinflammatory 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-B activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-␣ and IKK-␤-dependent signaling to NF-B. N1L associated with several components of the multisubunit I-B 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-B 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-B and IRF3 signaling pathways.

Detection of microbes by TLRs on the plasma membrane leads to the induction of pro-inflammatory 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, while endosomal TLR4 signaling to IRF3 via TRIF requires the TIR sorting adaptor TRAM. Similar to TLR4 homodimers, TLR2 heterodimers can also induce both pro-inflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, while endosomal IRF activation by TLR2 requires MyD88. However whether, like TLR4, TLR2 requires a sorting adaptor for endosomal signaling was unclear. Here 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.

Hypothermia was studied 5 min before, and 30 and 60 min after intraperitoneal administration of ethanol (3 g/kg) in 20 inbred strains of mice. Ethanol was given daily for 8 days, and temperatures were taken on Days 1, 3, 5, and 8. Tolerance was indexed by the reduction in hypothermia over days. There were large strain differences in baseline temperature, the hypothermic effect of ethanol, and in development of tolerance to hypothermia. Some strains of mice (DBA/1J, DBA/2N, MA/MyJ, and PL/J) did not develop tolerance to the hypothermic effect of ethanol. Initial sensitivity to the hypothermic effect of ethanol was significantly genetically correlated with tolerance development, indicating control of these responses by common genes. Ethanol-induced changes in activity and ataxia, as well as blood ethanol concentrations, were also assessed. Although there were significant strain differences in activity reduction, ataxia, blood-ethanol concentrations, and changes in these parameters during the course of chronic treatment, none of these variables could explain the genetic differences in hypothermic sensitivity and tolerance.