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: 10014186
• 负责人: Sonja Best
• 金额: $ 232.18万
• 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/10014186
• 关键词: Adaptor Signaling Protein; Affect; Amino Acids; Animal Model; Antigen Presentation; Antiviral Agents; Antiviral Response; Area; Arthropods; Automobile Driving; B-Cell Activation; Binding; Biology; Capsid; Categories; Cell Death; Cell Survival; Cell physiology; Cells; Central Nervous System Infections; Cessation of life; Characteristics; Cleaved cell; Clinical; Complex; Culicidae; Dendritic Cells; Dengue Virus; Development; Disease; Disease Outbreaks; Ebola virus; Endoplasmic Reticulum; Event; Evolution; Family; Filovirus; Flavivirus; Flavivirus Infections; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Host resistance; Hour; Human; Immune Evasion; Immune response; Immune system; Immunologics; Infection; Inflammation Mediators; Inflammatory Response; Innate Immune Response; Interferon Type I; Interferons; Intervention; Janus kinase; Japanese encephalitis virus; Laboratories; Langat virus; Lead; Ligation; Link; Liver; LoxP-flanked allele; Macaca mulatta; Membrane; Methyltransferase; Modeling; Mus; NF-kappa B; National Institute of Allergy and Infectious Disease; Natural Immunity; Nonstructural Protein; Nucleic Acids; Open Reading Frames; Pathogenesis; Pathway interactions; Pattern recognition receptor; Peptide Hydrolases; Polyproteins; Predisposition; Prevention; Proteins; RNA; RNA Helicase; RNA Processing; RNA Viruses; RNA-Directed RNA Polymerase; Recovery; Regulation; Research; Resistance; Retroviridae; Role; Signal Transduction; Signal Transduction Pathway; Site; Spleen; Structure; System; T-Lymphocyte; TNF gene; TRIM Gene; TRIM Motif; TRIM5 gene; Therapeutic; Tick-Borne Encephalitis; Tick-Borne Encephalitis Virus; Ticks; Toll-like receptors; Transcriptional Activation; Transducers; Translating; Tretinoin; Ubiquitination; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Nonstructural Proteins; Viral Pathogenesis; Viral Physiology; Virus; Virus Diseases; Virus Replication; West Nile virus; Work; Yellow Fever; Yellow fever virus; Zika Virus; adaptive immune response; base; burden of illness; cell type; chemokine; cytokine; human pathogen; immune activation; improved; in vivo; macrophage; member; mosquito-borne; mouse model; new therapeutic target; novel; pathogen; pathogenic virus; pressure; prevent; programs; response; single cell technology; success; therapeutic target; transcription factor; vector-borne; viral RNA; virus host interaction

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), tick-borne encephalitis virus (TBEV) and most recently Zika virus (ZIKV) that cause infections of the central nervous system. 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 more 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. Achieving these goals will significantly improve our understanding of how viruses emerge and cause disease in humans, as well as identify therapeutic targets for intervention. One major area of research is determining how genes induced by type I interferon, called ISGs, confer virus-specific protection. We have found a surprising new role for TRIM proteins in flavivirus-specific host protection. Tripartite motif-containing protein 5 (TRIM5) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5 is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we demonstrated that both human and rhesus macaque TRIM5 suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5 suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5 contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5 possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern. are now using new animal models to determine the role of these proteins in vivo and to determine if selective pressure from TRIM proteins is sufficient to drive flavivirus evolution and emergence. A second emphasis in the lab is the understanding of cell-type specific RLR signaling in host resistance to virus infection. We are using our floxed mouse model of conditional deletion of the MAVS gene. MAVS is an essential adaptor protein that connects the ligation of RIG-I-like RNA helicases by viral RNA to the expression of type I IFN. The conditional deletion of MAVS (MAVSfl/fl) enables selective depletion of this pathway in specific cell types such as macrophages and dendritic cells. Although we most commonly study flaviviruses, the unprecedented 2013-2016 outbreak of Ebola virus (EBOV) resulted in over 11,300 human deaths and necessitated additional work into host responses and so we applied EBOV to this mouse model. We applied a systems approach to MAVS-/- mice infected with either wild-type or mouse-adapted EBOV which revealed how MAVS controls EBOV replication through the expression of IFN, regulation of inflammatory responses in the spleen, and prevention of cell death in the liver. We are now using single-cell technologies to determine how RLR-MAVS signaling orchestrates protective antiviral responses in vivo.

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），以及引起中枢神经系统感染的日本脑炎病毒（JEV）、西尼罗河病毒（WNV）、蜱传脑炎病毒（TBEV）和最近出现的寨卡病毒（ZIKV）。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。实现这些目标将显着提高我们对病毒如何出现并导致人类疾病的理解，并确定干预的治疗靶点。 研究的一个主要领域是确定 I 型干扰素诱导的基因（称为 ISG）如何赋予病毒特异性保护。我们发现 TRIM 蛋白在黄病毒特异性宿主保护中具有令人惊讶的新作用。含三联基序的蛋白 5 (TRIM5) 是一种细胞抗病毒限制因子，可防止逆转录病毒复制的早期事件。由于与衣壳晶格高度特异性的相互作用，TRIM5 的活性被认为仅限于逆转录病毒。与目前的认识相反，我们证明人类和恒河猴 TRIM5 都能抑制特定黄病毒的复制。蜱传脑炎复合体中的多种病毒对 TRIM5 依赖性限制敏感，但蚊媒黄病毒，包括黄热病、登革热和寨卡病毒，具有抵抗力。 TRIM5 通过与病毒蛋白酶 NS2B/3 结合，促进其 K48 连接的泛素化和蛋白酶体降解来抑制复制。重要的是，TRIM5 有助于 IFN-I 对人类细胞中敏感黄病毒的抗病毒功能。因此，TRIM5在识别不同病毒家族方面具有显着的可塑性，有可能影响人类对全球关注的新兴黄病毒的易感性。现在正在使用新的动物模型来确定这些蛋白质在体内的作用，并确定 TRIM 蛋白质的选择压力是否足以驱动黄病毒的进化和出现。 实验室的第二个重点是了解宿主抵抗病毒感染的细胞类型特异性 RLR 信号传导。我们正在使用条件删除 MAVS 基因的 floxed 小鼠模型。 MAVS 是一种重要的衔接蛋白，它将病毒 RNA 的 RIG-I 样 RNA 解旋酶连接与 I 型 IFN 的表达连接起来。 MAVS (MAVSfl/fl) 的条件删除使得特定细胞类型（例如巨噬细胞和树突细胞）中的该通路选择性缺失。尽管我们最常研究黄病毒，但 2013 年至 2016 年史无前例的埃博拉病毒 (EBOV) 爆发导致 11,300 多人死亡，需要对宿主反应进行额外的研究，因此我们将 EBOV 应用于该小鼠模型。我们对感染野生型或小鼠适应型 EBOV 的 MAVS-/- 小鼠应用了系统方法，揭示了 MAVS 如何通过 IFN 的表达、调节脾脏炎症反应以及预防肝脏细胞死亡来控制 EBOV 复制。我们现在正在使用单细胞技术来确定 RLR-MAVS 信号如何协调体内保护性抗病毒反应。