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

宿主的先天免疫反应在病毒感染后几个小时内被触发。作为一个整体，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(PRRs)识别，包括Toll样受体(TLRs)和维甲酸诱导基因(RIG)样RNA解旋酶(RLHs)。通常由病毒核酸连接这些PRR，最终激活多个转录因子，这些转录因子协同驱动细胞因子和趋化因子的表达，这些细胞因子和趋化因子是固有反应的特征。核因子-kappaB和干扰素调节因子是特别重要的转录因子，负责诱导I型干扰素和肿瘤坏死因子α等炎症介质的产生。干扰素α/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-信号转导和转录激活(JAK-STAT)途径表达干扰素刺激基因(ISGs)。ISG的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，这些都可以直接影响病毒的复制。ISG在树突状细胞和巨噬细胞等免疫系统细胞中的表达对抗原提呈和T、B细胞的激活至关重要，从而影响获得性免疫反应的质量和最终的病毒清除。为了促进传播，致病病毒已经进化出一种机制，通过对抗这些信号转导途径来抑制宿主的先天免疫。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的发病机制以及开发有效的疫苗是至关重要的。 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感染的主要模型。黄病毒基本上分布在全球，对人类来说是一个巨大的疾病负担，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由节肢动物传播、由蚊子或扁虱传播这一事实有关。这一群体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)、森林脑炎病毒(TBEV)和最近的寨卡病毒(ZIKV)。这些病毒被列为NIAID A、B和C类病原体，用于研究它们的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架；由此产生的多蛋白被切割成至少10个蛋白质，包括3个结构蛋白(衣壳C、膜M，来源于前体Prem和包膜E)和7个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。病毒复制与来自宿主细胞内质网的修饰膜有关。NS5是黄病毒中最大、最保守的蛋白质，含有约900个氨基酸。它编码甲基转移酶(MTase)和RNA依赖的RNA聚合酶(RdRP)，并与NS3(病毒蛋白水解酶)结合形成病毒复制复合体的功能单位。尽管这些病原体造成了广泛且往往严重的感染，但疫苗只适用于少数几种病毒(YFV、JEV和TBEV)，并且没有治疗任何黄病毒引起的临床感染的治疗方法。 I型干扰素对黄病毒感染的康复至关重要，并已被用作潜在的治疗方法，尽管取得的成功有限。这可能是因为观察到，到目前为止所研究的所有黄病毒都通过抑制JAK-STAT信号转导来拮抗干扰素依赖的反应。我们确定NS5是由黄病毒编码的主要干扰素拮抗剂，最初使用兰加特病毒(LGTV；黄病毒TBEV复合体的成员)，最近使用WNV。虽然其他NS蛋白有助于抑制JAK-STAT信号，但我们实验室和其他人的研究表明，NS5是迄今为止所有载体传播的黄病毒编码的最有效的干扰素拮抗剂蛋白。因此，确定NS5阻碍信号转导的机制(S)对于了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ISGs的功能来了解干扰素抗病毒作用的潜在机制是重要的。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反应的发展和病毒致病机制中的作用是至关重要的。实现这些目标将大大提高我们对病毒如何出现并在人类中引发疾病的理解，以及确定干预的治疗目标。 我们今年工作的主要进展是与Adolfo Garcia-Sastre博士的实验室合作，确定寨卡病毒的NS5是I型干扰素信号的拮抗剂。寨卡病毒的NS5结合并降解来自人类和非人类灵长类动物的STAT2，而不是小鼠，这可能解释了为什么寨卡病毒感染的小鼠模型必须缺乏干扰素信号才能观察疾病。我们还注意到寨卡病毒NS5降解STAT2的策略与登革热病毒相似，但不同于其近亲海绵病毒。然而，与登革热病毒不同的是，寨卡病毒不利用E3泛素连接酶UBR4来诱导降解，这表明还有另一种机制尚未确定。

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. The major advance in our work this year was a collaboration with Dr. Adolfo Garcia-Sastre's laboratory resulting in the identification of NS5 from Zika virus as an antagonist of type I IFN signaling. NS5 from Zika virus binds and degrades STAT2 from humans and non-human primates, but not mice, which may explain why mouse models of Zika virus infection have to be deficient in IFN signaling in order to observe disease. We also noted that the strategy of Zika virus NS5 to degrade STAT2 is similar to that of dengue virus but not its closer relative called Spondweni virus.However, unlike dengue virus, Zika virus did not utilize the E3 ubiquitin ligase UBR4 to induce degradation suggesting that there is another mechanism that is yet to be identified.