Antagonism of Innate Immunity By Picornaviruses

描述(申请人提供)：所有细胞都具有对各种应激反应的遗传程序，如饥饿、温度、辐射和感染。这些进化的程序可以识别外来核酸和其他微生物产物，并对它们做出反应，以抵御细胞入侵。其中一个程序由先天性免疫防御系统组成，这种免疫系统由病毒感染激活，从而导致干扰素(干扰素)的合成。干扰素通过诱导干扰素刺激基因(ISGs)的表达来诱导抗病毒状态。IFN对抗病毒反应至关重要，但我们不知道编码ISG产物的1000多个基因如何建立抗病毒状态。目前对ISGs的作用机制及其靶向性知之甚少。这项应用中的实验利用一种特性良好的人类病原体--脊髓灰质炎病毒--来鉴定抑制ISG产物并研究它们的作用机制。的能力 脊髓灰质炎病毒在用干扰素处理的培养细胞中复制的能力可通过病毒2Apro蛋白酶中的单个氨基酸变化来消除。将2APro编码序列插入到干扰素敏感型脑心肌炎病毒(EMCV)的基因组中，使其能够在干扰素存在的情况下复制。我们的假设是，病毒2Apro蛋白拮抗一个或多个ISG产物的抗病毒活性。为了解决这一假设，我们将首先确定抑制脊髓灰质炎病毒复制的ISG产品及其作用机制(目标1)。我们将确定2Apro蛋白是否拮抗这些ISG产物的活性，并确定其机制。将表达脊髓灰质炎病毒2APro的重组EMCV在高水平的干扰素中传代，可筛选出对干扰素具有较高抗性的病毒突变体。负责这种表型的突变将被识别，并将确定它们在细胞因子存在时增强复制的机制。通过ISG反应保护小鼠的非神经细胞免受脊髓灰质炎病毒的感染。然而，脊髓灰质炎病毒在大脑和脊髓中复制良好，导致肌肉瘫痪。解释这些观察结果的一个假设是，神经元组织不会产生保护性的ISG反应，从而允许脊髓灰质炎病毒复制。在人神经母细胞瘤细胞系SK-N-SH中复制对干扰素敏感的脊髓灰质炎病毒2Apro突变体Y88L对干扰素相对不敏感，提示这些细胞可以作为一个模型来理解为什么中枢神经系统(CNS)的干扰素反应不会削弱脊髓灰质炎病毒的复制。在目标2中，我们将阐明HeLa和SK-N-SH细胞在抗病毒状态方面的差异。我们将确定在AIM 1中鉴定的ISG产物是否在SK-N-SH细胞中被诱导以响应病毒感染。如果ISG产物没有被诱导，我们将确定它们的过度表达是否保护神经细胞免受病毒感染。拟议的实验结果将确定抑制性ISGs及其作用机制。这一信息将为研究干扰素诱导的抗病毒状态提供新的见解，并可能为肠道病毒治疗提供新的靶点。

DESCRIPTION (provided by applicant): All cells possess genetic programs that respond to various stresses, such as starvation, temperature, irradiation, and infection. Those programs that have evolved to repel cellular invaders recognize and respond to alien nucleic acids and other microbial products. One such program consists of innate immune defenses, which are activated by viral infections, leading to the synthesis of interferon (IFN). IFNs induce an antivirl state by inducing the expression of IFN-stimulated genes (ISGs). IFNs are vital to the antiviral response, yet we do not understand how the 1000-plus genes encoding ISG products establish an antiviral state. There is little known about the mechanisms of action of ISGs, and their target specificity. Experiments in this application utilize a well-characterized human pathogen, poliovirus, to identify inhibitory ISG products and study their mechanism of action. The ability of poliovirus to replicate in cultured cells treated with IFN is abolished by a single amino acid change in the viral 2Apro proteinase. Insertion of the 2Apro coding sequence into the genome of the IFN-sensitive encephalomyocarditis virus (EMCV) confers the ability to replicate in the presence of IFN. Our hypothesis is that the viral 2Apro protein antagonizes the antiviral activity of one or more ISG products. To address this hypothesis, we will first identify ISG products that inhibit poliovirus replication and their mechanism of action (aim 1). We will determine if the 2Apro protein antagonizes the activity of these ISG products, and identify the mechanism. Passage of recombinant EMCV expressing poliovirus 2Apro in high levels of IFN leads to selection of viral mutants with higher resistance to IFN. Mutations responsible for this phenotype will be identified, and the mechanisms by which they enhance replication in the presence of the cytokine will be determined. Non-neuronal cells of mice are protected from poliovirus infection by the ISG response. However, poliovirus replicates well in the brain and spinal cord, leading to muscle paralysis. A hypothesis to explain these observations is that neuronal tissues do not mount a protective ISG response, allowing poliovirus replication. Replication of the IFN-sensitive poliovirus 2Apro mutant Y88L in the human neuroblastoma cell line SK-N-SH is relatively insensitive to IFN, suggesting that these cells may be used as a model for understanding why the IFN response in the central nervous system (CNS) does not impair poliovirus replication. In aim 2 we will elucidate the differences in the antiviral state between HeLa and SK-N-SH cells. We will determine if the ISG products identified in aim 1 are induced in SK-N-SH cells in response to viral infection. If the ISG products are not induced we will determine if their overexpression protects the neuronal cells from viral infection. The results of the proposed experiments will identify inhibitory ISGs and their mechanisms of action. This information will provide new insights into the IFN-induced antiviral state, and may provide novel targets for enteroviral therapeutics.