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.     

NFATc1 Mediates Toll-Like Receptor-Independent Innate Immune Responses during Trypanosoma cruzi Infection

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88 ^−/− Trif ^−/− mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-γ was normally induced in T. cruzi-infected Myd88 ^−/− Trif ^−/− innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca²⁺ level. Furthermore, T. cruzi-induced IFN-γ expression was blocked by inhibition of Ca²⁺ signaling. NFATc1, which plays a pivotal role in Ca²⁺ signaling in lymphocytes, was activated in T. cruzi-infected Myd88^−/−Trif^−/− innate immune cells. T. cruzi-infected Nfatc1 ^−/− fetal liver DCs were impaired in IFN-γ production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.     

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.     

Negative Regulation of Melanoma Differentiation-associated Gene 5 (MDA5)-dependent Antiviral Innate Immune Responses by Arf-like Protein 5B

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon β promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5.     

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．     

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.     

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.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

Innate Responses Induced by Whole Inactivated Virus or Subunit Influenza Vaccines in Cultured Dendritic Cells Correlate with Immune Responses In Vivo

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.     

Inhibition of Innate Immune Responses Is Key to Pathogenesis by Arenaviruses

Mammalian arenaviruses are zoonotic viruses that cause asymptomatic, persistent infections in their rodent hosts but can lead to severe and lethal hemorrhagic fever with bleeding and multiorgan failure in human patients. Lassa virus (LASV), for example, is endemic in several West African countries, where it is responsible for an estimated 500,000 infections and 5,000 deaths annually. There are currently no FDA-licensed therapeutics or vaccines available to combat arenavirus infection. A hallmark of arenavirus infection (e.g., LASV) is general immunosuppression that contributes to high viremia. Here, we discuss the early host immune responses to arenavirus infection and the recently discovered molecular mechanisms that enable pathogenic viruses to suppress host immune recognition and to contribute to the high degree of virulence. We also directly compare the innate immune evasion mechanisms between arenaviruses and other hemorrhagic fever-causing viruses, such as Ebola, Marburg, Dengue, and hantaviruses. A better understanding of the immunosuppression and immune evasion strategies of these deadly viruses may guide the development of novel preventative and therapeutic options.     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities

ATP-dependent chromatin remodeling activities function to manipulate chromatin structure during gene regulation. One of the ways in which they do this is by altering the positions of nucleosomes along DNA. Here we provide support for the ability of these complexes to move nucleosomes into positions in which DNA is unraveled from one edge. This is expected to result in the loss of histone-DNA contacts that are important for retention of one H2A/H2B dimer within the nucleosome. Consistent with this we find that several chromatin remodeling complexes are capable of catalyzing the exchange of H2A/H2B dimers between chromatin fragments in an ATP-dependent reaction. This provides eukaryotes with additional means by which they may manipulate chromatin structure.