痘病毒介导的NF-κB信号通路的调控

痘病毒是人和多种动物痘病的病原体,作为嗜上皮型的DNA病毒,在进化过程中,痘病毒特定的基因产物作用于机体免疫调控系统,调节免疫反应的进程和作用方式,进而完成其感染细胞、复制和繁殖的生物学过程。NF-κB信号通路是机体免疫系统重要的信号转导调节系统,各种痘病毒采用自身特殊的策略作用于这一免疫调节的重要靶系统,应对机体对其免疫清除和免疫反应。对痘病毒介导的宿主免疫调节的深入研究有助于研发新型疫苗和治疗性制剂。本文对近十年来各种痘病毒编码的多种目标蛋白参与宿主NF-κB信号通路调控的分子机制进行综述。     

A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB

The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.     

Metalloprotease type III effectors that specifically cleave JNK and NF-κB

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.     

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.     

Varicella-zoster virus inhibition of the NF-κB pathway during infection of human dendritic cells: role for open reading frame 61 as a modulator of NF-κB activity

Dendritic cells (DC) are antigen-presenting cells essential for initiating primary immune responses and therefore an ideal target for viral immune evasion. Varicella-zoster virus (VZV) can productively infect immature human DCs and impair their function as immune effectors by inhibiting their maturation, as evidenced by the expression modulation of functionally important cell surface immune molecules CD80, CD86, CD83, and major histocompatibility complex I. The NF-κB pathway largely regulates the expression of these immune molecules, and therefore we sought to determine whether VZV infection of DCs modulates the NF-κB pathway. Nuclear localization of NF-κB p50 and p65 indicates pathway activation; however, immunofluorescence studies revealed cytoplasmic retention of these NF-κB subunits in VZV-infected DCs. Western blotting revealed phosphorylation of the inhibitor of κBα (IκBα) in VZV-infected DCs, indicating that the pathway is active at this point. We conclude that VZV infection of DC inhibits the NF-κB pathway following protein phosphorylation but before the translocation of NF-κB subunits into the nucleus. An NF-κB reporter assay identified VZV open reading frame 61 (ORF61) as an inhibitor of tumor necrosis factor alpha-induced NF-κB reporter activity. Mutational analysis of ORF61 identified the E3 ubiquitin ligase domain as a region required for NF-κB pathway inhibition. In summary, we provide evidence that VZV inhibits the NF-κB signaling pathway in human DCs and that the E3 ubiquitin ligase domain of ORF61 is required to modulate this pathway. Thus, this work identifies a mechanism by which VZV modulates host immune function.     

The intricate interplay between RNA viruses and NF-κB

RNA viruses have rapidly evolving genomes which often allow cross-species transmission and frequently generate new virus variants with altered pathogenic properties. Therefore infections by RNA viruses are a major threat to human health. The infected host cell detects trace amounts of viral RNA and the last years have revealed common principles in the biochemical mechanisms leading to signal amplification that is required for mounting of a powerful antiviral response. Components of the RNA sensing and signaling machinery such as RIG-I-like proteins, MAVS and the inflammasome inducibly form large oligomers or even fibers that exhibit hallmarks of prions. Following a nucleation event triggered by detection of viral RNA, these energetically favorable and irreversible polymerization events trigger signaling cascades leading to the induction of antiviral and inflammatory responses, mediated by interferon and NF-κB pathways. Viruses have evolved sophisticated strategies to manipulate these host cell signaling pathways in order to ensure their replication. We will discuss at the examples of influenza and HTLV-1 viruses how a fascinating diversity of biochemical mechanisms is employed by viral proteins to control the NF-κB pathway at all levels.     

The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (ie1), WSSV303 and WSSV371

The Toll-like receptor (TLR)-mediated NF-κB pathway is essential for defending against viruses in insects and mammals. Viruses also develop strategies to utilize this pathway to benefit their infection and replication in mammal hosts. In invertebrates, the TLR-mediated NF-κB pathway has only been well-studied in insects and has been demonstrated to be important in antiviral responses. However, there are few reports of interactions between viruses and the TLR-mediated NF-κB pathway in invertebrate hosts. Here, we studied Litopenaeus vannamei Pelle, which is the central regulator of the Toll pathway, and proposed that a similar TLR/MyD88/Tube/Pelle/TRAF6/NF-κB cascade may exist in shrimp for immune gene regulation. After performing genome-wild analysis of white spot syndrome virus (WSSV) encoded proteins, we found that WSSV449 shows 15.7-19.4% identity to Tube, which is an important component of the insect Toll pathway. We further found that WSSV449 activated promoters of Toll pathway-controlled antimicrobial peptide genes, indicating WSSV449 has a similar function to host Tube in activating the NF-κB pathway. We suspected that WSSV449 activated the Toll-mediated NF-κB pathway for regulating viral gene expression. To test this hypothesis, we analyzed the promoters of viral genes and found 40 promoters that possess NF-κB binding sites. A promoter screen showed that the promoter activities of WSSV069 (ie1), WSSV303 and WSSV371 can be highly induced by the shrimp NF-κB family protein LvDorsal. WSSV449 also induced these three viral promoter activities by activating the NF-κB pathway. To our knowledge, this is the first report of a virus that encodes a protein similar to the Toll pathway component Tube to upregulate gene expression in the invertebrate host.     

7-ketocholesterol induces apoptosis in differentiated PC12 cells via reactive oxygen species-dependent activation of NF-κB and Akt pathways

Cholesterol oxidation products formed under the enhanced oxidative stress in the brain are suggested to induce neuronal cell death. However, it is still unknown whether oxysterol-induced apoptosis in neuronal cells is mediated by Akt and NF-κB pathways. We assessed the apoptotic effect of 7-ketocholesterol against differentiated PC12 cells in relation to activation of the reactive oxygen species-dependent nuclear factor (NF)-κB, which is mediated by the Akt pathway. 7-Ketocholesterol induced a decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic Bax levels, cytochrome c release, caspase-3 activation and upregulation of p53. 7-Ketocholesterol induced an increase in phosphorylated inhibitory κB-α, NF-κB p65 and NF-κB p50 levels, binding of NF-κB p65 to DNA, and activation of Akt. Treatment with Bay 11-7085 (an inhibitor of NF-κB activation) and oxidant scavengers, including N-acetylcysteine, prevented the 7-ketocholesterol-induced formation of reactive oxygen species, activation of NF-κB, Akt and apoptosis-related proteins, and cell death. Results from this study suggest that 7-ketocholesterol may exert an apoptotic effect against PC12 cells by inducing activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell death pathway, leading to activation of caspases, via the reactive oxygen species-dependent activation of NF-κB, which is mediated by the Akt pathway.     

TNFR1 signaling kinetics: Spatiotemporal control of three phases of IKK activation by posttranslational modification

TNFα is a pleotropic cytokine that plays a central role in the inflammatory response by activating the NF-κB signaling pathway, and is targeted in a range of chronic inflammatory diseases, underscoring the therapeutic importance of understanding its underlying molecular mechanisms. Although K63-linked ubiquitination of RIP1 by TRAF2/5 and cIAP1/2 was thought to serve as a scaffold to activate the NF-κB pathway, the recent accumulation of conflicting results has challenged the necessity of these proteins in NF-κB activation. In addition, several serine/threonine kinases have been implicated in TNFα-induced IKK activation; however, the targeted disruption of these kinases had no effect on transient IKK activation. The recent discovery of RIP1-dependent and -independent activation of the early and delayed phases of IKK and TRAF2 phosphorylation-dependent activation of the prolonged phase of IKK offers a reconciliatory model for the interpretation of contradictory results in the field. Notably, the TNFα-induced inflammatory response is not exclusively controlled by the NF-κB pathway but is subject to regulatory crosstalk between NF-κB and other context-dependent pathways. Thus further elucidation of these spatiotemporally-coordinated signaling mechanisms has the potential to provide novel molecular targets and therapeutic strategies for NF-κB intervention.     

Tuning NF-κB activity: A touch of COMMD proteins

NF-κB is an important regulator of immunity and inflammation, and its activation pathway has been studied extensively. The mechanisms that downregulate the activity of NF-κB have also received a lot of attention, particularly since its activity needs to be terminated to prevent chronic inflammation and subsequent tissue damage. The COMMD family has been identified as a new group of proteins involved in NF-κB termination. All ten COMMD members share the structurally conserved carboxy-terminal motif, the COMM domain, and are ubiquitously expressed. They seem to play distinct and non-redundant roles in various physiological processes, including NF-κB signaling. In this review, we describe the mechanisms and proteins involved in the termination of canonical NF-κB signaling, with a specific focus on the role of the COMMD family in the down-modulation of NF-κB.     

Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an α-methylene γ-lactone group and/or an α-bromo ketone group were synthesized and evaluated for their inhibitory effects on the NF-κB-dependent gene expression and signaling pathway. Our present study reveals that eudesmane-type α-methylene γ-lactones and α-bromo ketones inhibit multiple steps in the NF-κB signaling pathway induced by IL-1α and TNF-α.     

The C11R Gene, Which Encodes the Vaccinia Virus Growth Factor, Is Partially Responsible for MVA-Induced NF-κB and ERK2 Activation

MVA is an attenuated strain of vaccinia virus (VACV) that is a popular vaccine vector. MVA infection activates NF-κB. For 293T cells, it is known that MVA early gene expression activates extracellular signal-regulated kinase 2 (ERK2), resulting in NF-κB activation. However, other viral and cellular mechanisms responsible for this event are ill defined. The data presented here show that the epidermal growth factor receptor (EGFR) is at least one apical trigger in this pathway: ERK2 and NF-κB activation was diminished when MVA infections occurred in cells devoid of the EGFR (CHO K1 cells) or in the presence of a drug that inhibits EGFR activation (AG1478) in 293T cells. The expression of dominant negative Ras or Raf proteins still permitted NF-κB activation, suggesting that a nonclassical EGFR-based signal transduction pathway triggered ERK2-NF-κB activation. C11R is an early gene present in MVA and other orthopoxviruses. It encodes the soluble, secreted vaccinia virus growth factor (VGF), a protein that binds to and stimulates the EGFR. Here it was observed that NF-κB was activated in 293T cells transfected with a plasmid encoding the C11R gene. Silencing by small interfering RNA (siRNA) or deletion of the C11R gene (MVAΔC11R) reduced both MVA-induced ERK2 and NF-κB activation in 293T cells or the keratinocyte line Hacat, suggesting that this mechanism of MVA-induced NF-κB activation may be common for several cell types.