Virus-Host Interactions: Induction and Evasion of Host Innate Immunity

病毒与宿主的相互作用：宿主先天免疫的诱导和逃避

• 批准号: 8556030
• 负责人: Sonja Best
• 金额: $ 97.05万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556030
• 关键词: Affect; Amino Acids; Animal Model; Antigen Presentation; Antiviral Agents; Antiviral Response; Arthropods; Automobile Driving; B-Cell Activation; Biology; Bone Marrow; Capsid; Categories; Cell Maturation; Cell Survival; Cell physiology; Cells; Characteristics; Cleaved cell; Clinical; Complex; Culicidae; Dendritic Cells; Dengue Virus; Development; Disease; Encephalitis; Endoplasmic Reticulum; Flavivirus; Flavivirus Infections; Gene Expression; Genes; Genome; Goals; Host resistance; Hour; Human; IFNAR1 gene; Immune response; Immune system; Infection; Inflammation Mediators; Inflammatory; Interferon Receptor; Interferon Regulatory Factor 1; Interferon Type I; Interferon-beta; Interferons; Interleukin-12; Interleukin-6; Janus kinase; Japanese Encephalitis Viruses; Japanese encephalitis virus; Kinetics; Laboratories; Langat virus; Lead; Ligation; Lysosomes; Mediating; Membrane; Messenger RNA; Methyltransferase; Modeling; Mus; NF-kappa B; National Institute of Allergy and Infectious Disease; Natural Immunity; Nonstructural Protein; Nucleic Acids; Open Reading Frames; PTPN11 gene; Pathogenesis; Pathway interactions; Pattern recognition receptor; Peptide Hydrolases; Play; Polyproteins; Production; Proteins; RNA; RNA Helicase; RNA Processing; RNA-Directed RNA Polymerase; Recovery; Research; Role; Signal Transduction; Signal Transduction Pathway; Site; Specificity; TRIM Motif; Therapeutic; Tick-Borne Encephalitis Virus; Tick-Borne Encephalitis Viruses; Ticks; Toll-like receptors; Transcriptional Activation; Transducers; Translating; Tretinoin; Tumor Necrosis Factor-alpha; Vaccine Design; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Nonstructural Proteins; Viral Pathogenesis; Viral Physiology; Virus; Virus Diseases; Virus Replication; West Nile virus; Yellow fever virus; burden of illness; cell type; chemokine; cytokine; human TNF protein; immune activation; improved; interleukin-12 subunit p35; interleukin-12 subunit p40; mRNA Expression; macrophage; member; new therapeutic target; novel; novel therapeutics; nuclear factor 1; pathogen; programs; response; success; transcription factor; vaccine development; vector; virus host interaction; virus pathogenesis

The host innate immune response is triggered within hours of virus infection. As a whole, its function is to limit virus replication at local sites of infection and to orchestrate development of the adaptive immune response. Viruses are typically recognized by cellular pattern recognition receptors (PRRs), including toll-like receptors (TLRs) and the retinoic acid inducible gene (RIG)-like RNA helicases (RLHs). Ligation of these PRRs, often by viral nucleic acids, culminates in the activation of multiple transcription factors that cooperate in driving expression of cytokines and chemokines characteristic of the innate response. Nuclear factor-kappa B (NF-kappaB) and interferon (IFN) regulatory factors (IRFs) are particularly important transcription factors, responsible for induction of type I IFN (IFNalpha/beta), tumor necrosis factor alpha (TNFalpha) and other mediators of inflammation. IFNalpha/beta is central to the anti-viral response as it initiates its own transcriptional program resulting in expression of IFN-stimulated genes (ISGs) via the Janus kinase-signal transducer and activation of transcription (JAK-STAT) pathway. ISG expression influences many cellular processes including RNA processing, protein stability and cell viability that can directly affect virus replication. ISG expression in cells of the immune system such as dendritic cells (DCs) and macrophages is critical for antigen presentation and T- and B-cell activation, thus affecting the quality of the adaptive immune response and eventual virus clearance. To facilitate dissemination, pathogenic viruses have evolved mechanisms to suppress host innate immunity by antagonizing these signal transduction pathways. Hence, understanding the specific pathways by which viruses activate and evade innate immune responses is essential for understanding viral pathogenesis as well as for development of effective vaccines. To examine virus-host interactions that affect innate immunity, our laboratory utilizes flaviviruses as the primary model of infection. Flaviviruses have an essentially global distribution and represent a tremendous disease burden to humans, causing millions of infections annually. The success of flaviviruses as human pathogens is associated with the fact that they are arthropod-borne, transmitted by mosquitoes or ticks. Significant members of this group include dengue virus (DENV) and yellow fever virus (YFV) that cause hemorrhagic fevers, as well as Japanese encephalitis virus (JEV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) that cause severe encephalitides. These viruses are listed as NIAID category A, B and C pathogens for research into their basic biology and host response. The flavivirus single-stranded RNA genome is translated as one open reading frame; the resulting polyprotein is cleaved into at least ten proteins that include three structural (capsid C, membrane M, derived from the precursor preM and envelope E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Virus replication proceeds in association with modified membranes derived from the endoplasmic reticulum of host cells. NS5 is the largest and most conserved of the flavivirus proteins containing approximately 900 amino acids. It encodes a methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRP) and associates with NS3 (the viral protease) to form the functional unit of the viral replication complex. Despite the widespread and often severe infections caused by these pathogens, vaccines exist for only a few (YFV, JEV and TBEV) and no therapeutic exists to treat clinical infection caused by any flavivirus. Type I IFNs are essential to recovery from flavivirus infection and have been used clinically as potential therapeutics, albeit with limit success. This may be due to the observation that all flaviviruses examined to date antagonize IFN-dependent responses by suppressing JAK-STAT signal transduction. We identified NS5 as the major IFN antagonist encoded by flaviviruses, originally using Langat virus (LGTV; a member of the TBEV complex of flaviviruses) and most recently using WNV. Although other NS proteins contribute to suppression of JAK-STAT signaling, studies by our laboratory and others suggest that NS5 is the most potent of the IFN antagonist proteins encoded by all vector-borne flaviviruses examined thus far. Hence, determining the mechanism(s) by which NS5 impedes signaling is essential to understand flavivirus pathogenesis and may lead to new therapeutic targets. Furthermore, it is important to understand the mechanisms underlying the anti-viral effects of IFN by identifying the function of ISGs with anti-viral activity. Finally, it is essential to translate these findings to immunologically relevant cell types and animal models to understand the roles of induction and evasion of innate immunity in development of the adaptive immune response and in virus pathogenesis. Our progress this year includes the demonstration that the tripartite motif (TRIM) protein, TRIM79alpha is an ISG that specifically targets TBEV. TRIM79alpha restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein. NS5 degradation was specific to tick-borne flaviviruses as TRIM79alpha did not recognize NS5 from WNV or inhibit WNV replication. In the absence of TRIM79alpha, IFN-beta was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79alpha for TBEV reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses. We have also examined the effects of flavivirus infection of DC maturation. Maturation of DCs and their subsequent ability to initiate adaptive immune responses is coordinated by PRR ligation, transcription factor activation and type I IFN) production. Early activation of interferon regulatory factor-1 (IRF-1), a transcription factor, results in expression of broadly acting antiviral effector genes including interleukin-12 (IL-12), a cytokine that plays an essential role in bridging innate and adaptive immune responses. To understand the impact of tick-borne flaviviruses on DC responses, we examined the maturation profile and cytokine response of murine bone marrow-derived DCs infected with Langat virus (LGTV; a member of the TBEV serogroup). In response to PRR stimulation, LGTV infected DCs showed enhanced production of TNF-alpha and IL-6, but suppressed IL-12 compared to uninfected cells in the same culture or mock-infected controls. The differential modulation of IL-12 was due to reduced expression of IL-12p40 mRNA, but not that of IL-12p35. Kinetic analysis revealed that early IL-12p40 mRNA expression required IRF-1 whereas sustained gene expression was dependent on signaling through the IFN receptor (IFNAR). Examination of IRF-1 status revealed that LGTV or TBEV infection resulted in accelerated IRF-1 degradation. Moreover, DCs enriched from LGTV infected Ifnar-/- mice showed impaired IRF-1 nuclear localization. Irf-1-/- mice were highly susceptible to LGTV infection suggesting that IRF-1 antagonism contributes to virus pathogenesis. Thus, IRF-1 degradation is a novel mechanism that synergizes with the noted ability of flaviviruses to suppress IFNAR-dependent signaling, resulting in the orchestrated evasion of host innate immunity.

病毒感染后数小时内就会触发宿主先天免疫反应。总体而言，其功能是限制病毒在局部感染部位的复制并协调适应性免疫反应的发展。病毒通常由细胞模式识别受体 (PRR) 识别，包括 Toll 样受体 (TLR) 和视黄酸诱导基因 (RIG) 样 RNA 解旋酶 (RLH)。通常通过病毒核酸连接这些 PRR，最终导致多种转录因子的激活，这些转录因子协同驱动先天反应特征的细胞因子和趋化因子的表达。核因子-κ B (NF-κB) 和干扰素 (IFN) 调节因子 (IRF) 是特别重要的转录因子，负责诱导 I 型 IFN (IFNα/β)、肿瘤坏死因子 α (TNFα) 和其他炎症介质。 IFNα/β 是抗病毒反应的核心，因为它启动自己的转录程序，通过 Janus 激酶信号转导器和转录激活 (JAK-STAT) 途径表达 IFN 刺激基因 (ISG)。 ISG 表达影响许多细胞过程，包括 RNA 加工、蛋白质稳定性和细胞活力，可直接影响病毒复制。树突状细胞 (DC) 和巨噬细胞等免疫系统细胞中的 ISG 表达对于抗原呈递以及 T 和 B 细胞激活至关重要，从而影响适应性免疫反应的质量和最终的病毒清除。为了促进传播，病原病毒已经进化出通过拮抗这些信号转导途径来抑制宿主先天免疫的机制。因此，了解病毒激活和逃避先天免疫反应的具体途径对于了解病毒发病机制以及开发有效的疫苗至关重要。 为了检查影响先天免疫的病毒-宿主相互作用，我们的实验室利用黄病毒作为主要感染模型。黄病毒基本上在全球范围内分布，给人类带来巨大的疾病负担，每年造成数百万例感染。黄病毒作为人类病原体的成功与它们是节肢动物传播的、通过蚊子或蜱传播的事实有关。该组的重要成员包括引起出血热的登革热病毒（DENV）和黄热病病毒（YFV），以及引起严重脑炎的日本脑炎病毒（JEV）、西尼罗河病毒（WNV）和蜱传脑炎病毒（TBEV）。这些病毒被列为 NIAID A、B 和 C 类病原体，用于研究其基本生物学和宿主反应。黄病毒单链RNA基因组被翻译为一个开放阅读框；所得多蛋白被切割成至少十种蛋白质，其中包括三种结构蛋白（衣壳 C、膜 M、源自前体 preM 和包膜 E）和七种非结构蛋白（NS1、NS2A、NS2B、NS3、NS4A、NS4B 和 NS5）。病毒复制与源自宿主细胞内质网的修饰膜相关。 NS5 是最大、最保守的黄病毒蛋白，含有约 900 个氨基酸。它编码甲基转移酶 (MTase) 和 RNA 依赖性 RNA 聚合酶 (RdRP)，并与 NS3（病毒蛋白酶）结合形成病毒复制复合物的功能单元。尽管这些病原体引起的感染广泛且往往严重，但只有少数几种病原体（YFV、JEV 和 TBEV）存在疫苗，并且没有治疗方法可以治疗任何黄病毒引起的临床感染。 I 型干扰素对于从黄病毒感染中恢复至关重要，并且已在临床上用作潜在的治疗方法，尽管成功程度有限。这可能是由于观察到迄今为止检查的所有黄病毒都通过抑制 JAK-STAT 信号转导来拮抗 IFN 依赖性反应。我们确定 NS5 是由黄病毒编码的主要 IFN 拮抗剂，最初使用 Langat 病毒（LGTV；黄病毒 TBEV 复合体的成员），最近使用 WNV。尽管其他 NS 蛋白有助于抑制 JAK-STAT 信号传导，但我们实验室和其他实验室的研究表明，NS5 是迄今为止检查的所有载体传播黄病毒编码的最有效的 IFN 拮抗剂蛋白。 因此，确定 NS5 阻碍信号传导的机制对于了解黄病毒发病机制至关重要，并可能导致新的治疗靶点。此外，通过鉴定具有抗病毒活性的 ISG 的功能来了解 IFN 抗病毒作用的机制也很重要。最后，必须将这些发现转化为免疫学相关的细胞类型和动物模型，以了解先天免疫的诱导和逃避在适应性免疫反应的发展和病毒发病机制中的作用。 我们今年的进展包括证明三联基序 (TRIM) 蛋白 TRIM79alpha 是一种专门针对 TBEV 的 ISG。 TRIM79alpha 通过介导黄病毒 NS5 蛋白的溶酶体依赖性降解来限制 TBEV 复制。 NS5 降解是蜱传黄病毒所特有的，因为 TRIM79alpha 不能识别 WNV 中的 NS5 或抑制 WNV 复制。在缺乏 TRIM79α 的情况下，IFN-β 在抑制小鼠巨噬细胞的蜱传黄病毒感染方面效果较差，这凸显了单一病毒特异性 ISG 在建立抗病毒状态中的重要性。 TRIM79alpha 对 TBEV 的特异性揭示了先天 IFN 反应区分密切相关的黄病毒的非凡能力。 我们还研究了黄病毒感染对 DC 成熟的影响。 DC 的成熟及其随后启动适应性免疫反应的能力是通过 PRR 连接、转录因子激活和 I 型 IFN 的产生来协调的。干扰素调节因子-1 (IRF-1)（一种转录因子）的早期激活会导致广泛作用的抗病毒效应基因的表达，包括白细胞介素-12 (IL-12)，这是一种在桥接先天性和适应性免疫反应中发挥重要作用的细胞因子。为了了解蜱传黄病毒对 DC 反应的影响，我们检查了感染 Langat 病毒（LGTV；TBEV 血清群成员）的鼠骨髓来源 DC 的成熟特征和细胞因子反应。为了响应 PRR 刺激，与相同培养物或模拟感染对照中未感染的细胞相比，LGTV 感染的 DC 表现出 TNF-α 和 IL-6 的产生增强，但 IL-12 受到抑制。 IL-12 的差异调节是由于 IL-12p40 mRNA 表达减少所致，而非 IL-12p35 mRNA 表达减少所致。动力学分析表明，早期 IL-12p40 mRNA 表达需要 IRF-1，而持续基因表达则依赖于通过 IFN 受体 (IFNAR) 的信号传导。 IRF-1 状态检查显示 LGTV 或 TBEV 感染导致 IRF-1 加速降解。此外，从 LGTV 感染的 Ifnar-/- 小鼠中富集的 DC 表现出 IRF-1 核定位受损。 Irf-1-/- 小鼠对 LGTV 感染高度敏感，表明 IRF-1 拮抗作用有助于病毒发病机制。因此，IRF-1 降解是一种新机制，与黄病毒抑制 IFNAR 依赖性信号传导的显着能力协同作用，导致宿主先天免疫的精心策划的逃避。