TLS/FUS

Comment on: Tan AY, et al. Proc Natl Acad Sci USA 2012; 109:6030-5.     

人类冠状病毒调节宿主抗病毒天然免疫分子机制

SARS冠状病毒和正在全球流行的猪源H1N1型流感病毒等人类新发呼吸道病毒对人类生命健康构成严重威胁.人类重要呼吸道病毒与宿主抗病毒天然免疫的关系是近年来研究热点.SARS冠状病毒等很多RNA病毒能够编码某种蛋白质,抑制干扰素表达以及干扰素介导的抗病毒信号通路.人类冠状病毒木瓜样蛋白酶(papain-like protease,PLP)利用其自身去泛素化酶(DUB)活性,使干扰素表达通路中重要调节蛋白发生去泛素化,从而抑制干扰素信号传导.同时,PLP蛋白酶通过阻碍干扰素表达信号通路中最新发现的重要调节蛋白ERIS(也称MITA/STING)二聚化,使其失活并丧失激活干扰素通路的功能,这些发现对于阐明人类重要呼吸道病毒对宿主细胞抗病毒天然免疫反应的调节作用及其机制具有重要意义,为人类新发病毒致病机理、免疫防治以及抗病毒药物研究提供新的思路.     

Interferon Lambda Alleles Predict Innate Antiviral Immune Responses and Hepatitis C Virus Permissiveness

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Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses

Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.One of the most significant evolutionary features in eukaryotes is the appearance of a membrane system to separate enzymatic reactions and to provide scaffolds for signal transduction. The small genome of viruses requires that they use the host''s cellular machinery, especially host intracellular membranes, to assemble their replication complexes and to complete their replication cycle (1). Therefore, it is not surprising to find that the eukaryotic membrane system is also involved in antiviral responses (2–4).Subcellular organelles with extensive membrane systems include the endoplasmic reticulum (ER)¹, mitochondria, endosomes and peroxisomes. Despite their well-recognized functions in cell metabolism, these organelles and their related membranes have been identified in recent years as important innate immune platforms (5). The ER is a key organelle for maintaining cellular homeostasis. Several recent studies have linked the ER to antiviral immune responses and have elucidated the related mechanisms (6–10). The mitochondria serve mainly as the power plants of eukaryotic cells but also participate in numerous crucial cellular processes, such as calcium homeostasis, apoptosis and aging (11–13). In recent years, the mitochondria and mitochondrial-associated membranes (MAM) have also emerged as fundamental hubs for innate antiviral immunity. Several important antiviral immune-related proteins, such as VISA (also referred to as MAVS/CARDIF/IPS-1) and MITA (also referred to as STING), have been found to localize to the mitochondrial membrane (14–19). Peroxisomes are monolayer-membrane organelles present in nearly all types of human cells, with a particularly high abundance in hepatocytes and nephrocytes, which are involved in various oxidative enzymatic reactions. It has been reported that the peroxisomal-associated protein VISA induces a rapid interferon (IFN)-independent expression that provides short-term protection, whereas the mitochondrial-associated VISA activates an IFN-dependent signaling pathway with delayed kinetics (20). The peroxisomal VISA can activates IRF1-mediated IFN-λ production (21). The endosomes, although known as players in cellular endocytosis and vascular transport, have functions that extend to the antigen presentation of major histocompatibility complex (MHC) class I, MHC class II and CD1 molecules of the adaptive immune system, as well as pattern recognition of innate immune-related receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) (22, 23).Subcellular fractionation is an effective experimental strategy for isolating/enriching specific organelles. Compared with whole-cell-based proteomic analysis, combining the isolation of subcellular components with mass spectrometry-based proteomic analysis is advantageous not only for characterizing low abundance proteins but also for monitoring protein abundance changes at the organelle level. To systematically analyze the role of peroxisomal-related proteins in innate immune responses, we used a modified two-step gradient centrifugation method to enrich the peroxisomes from cells with or without SeV infection, followed by a quantitative proteomic analysis. A total of 2946 proteins were quantified among which 311 proteins were found to be significantly changed by SeV infection. A statistical enrichment test was used to reveal that 13 protein groups were changed significantly (p < 0.05) compared with the entire protein list. Cell cycle-related proteins and membrane attack complex (MAC)-related proteins were down-regulated, which may facilitate virus survival and replication in host cells. Luciferase reporter assays were performed to further screen for the significantly changed proteins that could affect SeV-induced activation of the type I IFN signaling pathway. Not only does our data provide new and unbiased protein-level information regarding viral infection processes, we also provide direct evidence for the involvement of two proteins (CALU and LGALS3BP) as potential negative regulators in the virus-triggered induction of the type I IFNs.     

Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background

Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.

Aim

To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.

Methodology and principal findings

Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4⁺ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.

Conclusions and significance

Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.     

Extrachromosomal Histone H2B Mediates Innate Antiviral Immune Responses Induced by Intracellular Double-Stranded DNA

Fragments of double-stranded DNA (dsDNA) forming a right-handed helical structure (B-DNA) stimulate cells to produce type I interferons (IFNs). While an adaptor molecule, IFN-β promoter stimulator 1 (IPS-1), mediates dsDNA-induced cellular signaling in human cells, the underlying molecular mechanism is not fully understood. Here, we demonstrate that the extrachromosomal histone H2B mediates innate antiviral immune responses in human cells. H2B physically interacts with IPS-1 through the association with a newly identified adaptor, CIAO (COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1), to transmit the cellular signaling for dsDNA but not immunostimulatory RNA. Extrachromosomal histone H2B was biologically crucial for cell-autonomous responses to protect against multiplication of DNA viruses but not an RNA virus. Thus, the present findings provide evidence indicating that the extrachromosomal histone H2B is engaged in the signaling pathway initiated by dsDNA to trigger antiviral innate immune responses.Fragments of nucleic acids derived from either infectious agents or host cells activate cell-autonomous responses to inhibit multiplication of certain viruses by inducing type I interferon (IFN) production (5). Such effects are more evident when double-stranded DNA (dsDNA) is transduced into the intracellular compartment by use of a transfection agent or electroporation method, suggesting that the DNA sensing system recognizes aberrant DNA fragments inside the cell (6, 21, 23). dsDNA forming a right-handed helical structure, i.e., B-DNA, has a greater ability to induce type I IFNs than Z-DNA, which has a left-handed zig-zag structure (6). dsDNA activates type I IFN production in a wide variety of cell types, including immune cells, such as dendritic cells and macrophages, and nonimmune cells, such as fibroblasts, epithelial cells, and thyroid cells (6, 23). Such effects of dsDNA were corroborated by the observation in mice deficient for DNase II, in which intracellular accumulation of undegraded DNA fragments resulted in hyperproduction of IFN-β, dysregulation of erythropoiesis, and symptoms resembling rheumatoid arthritis (12, 28). The loss-of-function mutation of the DNase I gene has been found in patients with systemic lupus erythematosus (SLE) and, in fact, DNase I^−/− mice manifest SLE-like symptoms with anti-DNA antibody (Ab) production (18, 27).The immunostimulatory property of dsDNA is quite similar to that of immunostimulatory RNA (isRNA), such as dsRNA and 5′-triphosphate RNA (2, 6). Indeed, the signaling pathways engaged by dsDNA in part are shared with those for isRNA. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) directly associate with isRNA and trigger signaling, while it has been demonstrated that RIG-I does not directly interact with dsDNA but mediates its signaling in a human hepatoma cell line, Huh7 (2). IFN-β promoter stimulator 1 (IPS-1, also known as mitochondrial antiviral signaling), mediates the downstream signaling induced by dsDNA or isRNA in humans, while IPS-1 solely mediates isRNA but not dsDNA signaling in mice (2, 6, 15, 22). In contrast, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi) are essential for dsDNA- or isRNA-induced type I IFN production in both humans and mice (2, 6). While examining distinct molecules involved in dsDNA-mediated but not isRNA-mediated upstream signaling, Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN regulatory factors [DAI]) was identified as a candidate cytosolic DNA sensor, at least in a mouse connective tissue cell line, L929, although its in vivo role was dispensable (7, 24, 26). Recently, a PYHIN family member, Absent in melanoma 2 (AIM2) protein, was shown to associate with an inflammasome signaling adaptor, apoptosis-associated speck-like protein containing a CARD (ASC), and to play a critical role for caspase 1 activation and interleukin-1β (IL-1β) secretion in response to dsDNA (1, 3, 4, 20).In the present study, we show that extrachromosomal histone H2B is responsible for the dsDNA-induced type I IFN production in human cells and for the innate immune response to DNA virus infection.     

Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.     

NL63冠状病毒木瓜样蛋白酶去泛素化酶活性和对宿主抗病毒天然免疫反应调节作用

引起人类呼吸道感染的冠状病毒已多达5种．冠状病毒与宿主相互作用决定了其致病性和免疫特性．冠状病毒感染后宿主会立即启动抗病毒天然免疫反应,而人类冠状病毒往往会编码特定蛋白逃逸或抑制宿主的天然免疫反应．NL63冠状病毒是一种新型人类冠状病毒,其非结构蛋白nsp3编码2个木瓜样蛋白酶(PLP)核心结构域PLP1和PLP2．前期研究发现,人类冠状病毒PLP2是一种病毒编码的去泛素化酶(DUB),但是对其DUB特性和功能还不清楚．研究发现,NL63冠状病毒PLP1和PLP2两个核心结构域中只有PLP2具有DUB活性,而且,PLP2的DUB活性对K48和K63连接的多聚泛素化修饰不表现明显特异性．同时,蛋白酶活性催化位点C1678和H1836突变后对其DUB活性有明显抑制作用,而蛋白酶活性催化位点D1849突变后对DUB活性无影响．其次,PLP2而非PLP1核心结构域能够明显抑制仙台病毒和重要信号蛋白(RIG-I、ERIS/STING/MITA)激活的干扰素表达,表明PLP2是一种冠状病毒编码的干扰素拮抗剂,而且PLP2的干扰素拮抗作用不完全依赖其蛋白酶活性．机制研究表明,PLP2能够与干扰素表达通路中的重要调节蛋白RIG-I和ERIS发生相互作用,通过对RIG-I和ERIS的去泛素化负调控宿主抗病毒天然免疫反应．此外,PLP2除利用DUB活性抑制干扰素表达外,很可能存在不依赖自身催化活性的其他组分共同抑制干扰素的产生．以上研究对阐明人类新发冠状病毒免疫和致病机理以及抗病毒药物研发具有重要参考价值．     

Paramyxovirus Activation and Inhibition of Innate Immune Responses

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.     

流感病毒先天免疫应答和先天免疫逃逸的机制

流感病毒引起人类和动物的呼吸道感染已是全世界严重的经济和公共卫生问题。在感染早期,流感病毒会导致机体的先天免疫信号被激活,起到防御、清除病毒以及辅助适应性免疫应答的作用。但在与宿主共进化的过程中,流感病毒形成了多种逃逸策略,主要是通过病毒自身蛋白质阻断宿主天然免疫通路,抑制干扰素和炎性因子的生成。基于现有的研究成果,本文针对流感病毒先天免疫应答和先天免疫逃逸的机制做一扼要综述,这有助于加强流感病毒抗原进化的监测、探索疫苗和抗病毒药物的合理靶标,为更好地预防和控制该病提供有效的策略。     

Staphylococcus aureus Alpha Toxin Suppresses Effective Innate and Adaptive Immune Responses in a Murine Dermonecrosis Model

An optimal host response against Staphylococcus aureus skin and soft tissue infections (SSTI) is dependent on IL-1β and IL-17 mediated abscess formation. Alpha toxin (AT), an essential virulence factor for SSTI, has been reported to damage tissue integrity; however its effect on the immune response has not been investigated. Here, we demonstrate that infection with USA300 AT isogenic mutant (Δhla), or passive immunization with an AT neutralizing mAb, 2A3, 24 h prior to infection with wild type USA300 (WT), resulted in dermonecrotic lesion size reduction, and robust neutrophil infiltration. Infiltration correlates with increase in proinflammatory cytokines and chemokines, as well as enhanced bacterial clearance relative to immunization with a negative control mAb. In addition, infection with Δhla, or with WT +2A3, resulted in an early influx of innate IL-17⁺γδT cells and a more rapid induction of an adaptive immune response as measured by Th1 and Th17 cell recruitment at the site of infection. These results are the first direct evidence of a role for AT in subverting the innate and adaptive immune responses during a S. aureus SSTI. Further, these effects of AT can be overcome with a high affinity anti-AT mAb resulting in a reduction in disease severity.     

Induction and Evasion of Innate Antiviral Responses by Hepatitis C Virus

Persistent hepatitis C virus infection is associated with progressive hepatic fibrosis and liver cancer. Acute infection evokes several distinct innate immune responses, but these are partially or completely countered by the virus. Hepatitis C virus proteins serve dual functions in replication and immune evasion, acting to disrupt cellular signaling pathways leading to interferon synthesis, subvert Jak-STAT signaling to limit expression of interferon-stimulated genes, and block antiviral activities of interferon-stimulated genes. The net effect is a multilayered evasion of innate immunity, which negatively influences the subsequent development of antigen-specific adaptive immunity, thereby contributing to virus persistence and resistance to therapy.     

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Background

Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.

Results

We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.

Conclusions

Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.