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: 8157105
• 负责人: Sonja Best
• 金额: $ 135.55万
• 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/8157105
• 关键词: Natural Immunity; 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), namely 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 NS5 from the virulent NY99 strain of WNV prevented pY-STAT1 accumulation, suppressed IFN-dependent gene expression, and rescued the growth of a highly IFN-sensitive virus (Newcastle disease virus) in the presence of IFN, suggesting that this protein can function as an efficient IFN antagonist. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of pY-STAT1. Mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered KUN NS5 an efficient inhibitor of pY-STAT1. Incorporation of this mutation into recombinant KUN resulted in 30-fold greater inhibition of JAK-STAT signaling than with the wild-type virus and enhanced KUN replication in the presence of IFN. Thus, a naturally occurring mutation is associated with the function of NS5 in IFN antagonism and may influence virulence of WNV field isolates.

宿主的先天免疫反应在病毒感染后数小时内被触发。总的来说，它的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，即toll样受体（TLR）和视黄酸诱导基因（RIG）样RNA解旋酶（RLH）。这些PRR的连接，通常通过病毒核酸，最终激活多个转录因子，这些转录因子在驱动先天反应特征性的细胞因子和趋化因子的表达中合作。核因子-κ B（NF-κ B）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（IFN α/β）、肿瘤坏死因子α（TNF α）和其它炎症介质。IFN α/β是抗病毒应答的核心，因为它启动其自身的转录程序，导致IFN刺激的基因（ISG）通过Janus激酶信号转导和转录激活（JAK-STAT）途径表达。 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。免疫系统细胞如树突状细胞（DC）和巨噬细胞中的ISG表达对于抗原呈递和T细胞和B细胞活化至关重要，从而影响适应性免疫应答的质量和最终的病毒清除。为了促进传播，病原性病毒进化出了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应答的特定途径对于理解病毒发病机制以及开发有效的疫苗至关重要。 为了研究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的感染模型。黄病毒具有基本上全球性的分布，并且对人类造成巨大的疾病负担，每年造成数百万人感染。黄病毒作为人类病原体的成功与它们是节肢动物传播的，由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，包括三种结构蛋白（衣壳C，膜M，来源于前体preM和包膜E）和七种非结构蛋白（NS 1，NS 2A，NS 2B，NS 3，NS 4A，NS 4 B和NS 5）。病毒复制与来自宿主细胞内质网的修饰膜相关。NS 5是最大和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NS 3（病毒蛋白酶）结合形成病毒复制复合物的功能单位。尽管由这些病原体引起的广泛且通常严重的感染，但仅存在用于少数（YFV、JEV和TBEV）的疫苗，并且不存在治疗由任何黄病毒引起的临床感染的治疗剂。 I型IFN对于从黄病毒感染中恢复是必不可少的，并且已在临床上用作潜在的治疗剂，尽管成功有限。这可能是由于观察到迄今为止检查的所有黄病毒通过抑制JAK-STAT信号转导来拮抗IFN依赖性应答。我们确定NS 5作为主要的IFN拮抗剂编码的黄病毒，最初使用Langat病毒（LGTV;一个成员的TBEV复杂的黄病毒），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人的研究表明，NS 5是迄今为止检查的所有媒介传播黄病毒编码的最有效的IFN拮抗剂蛋白。 因此，确定NS 5阻碍信号传导的机制对于理解黄病毒的发病机制至关重要，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，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应答的发展和病毒发病机制中的作用是至关重要的。 我们今年的进展包括证明来自WNV强毒株NY 99的NS 5阻止了pY-STAT 1的积累，抑制了IFN依赖性基因的表达，并在IFN存在下挽救了高度IFN敏感的病毒（纽卡斯尔病病毒）的生长，这表明这种蛋白质可以作为有效的IFN拮抗剂发挥作用。相反，来自昆津病毒（Kunjin virus，KUN）（WNV的一种天然减毒亚型）的NS 5是pY-STAT 1的弱抑制剂。KUN NS 5中的单个残基突变为WNV-NY 99 NS 5中的类似残基（S653 F）使得KUN NS 5成为pY-STAT 1的有效抑制剂。将该突变并入重组KUN中导致JAK-STAT信号传导的抑制比野生型病毒大30倍，并且在IFN存在下增强KUN复制。因此，自然发生的突变与NS 5在IFN拮抗作用中的功能相关，并可能影响WNV田间分离株的毒力。