Cardioviral Proteases and Comparative Genome Structure

这项调查的目的是探索和确定心脏病毒属与其他 微小核糖核酸病毒家族的成员，并利用心脏病毒的独特功能来检查 微小核糖核酸病毒的翻译、蛋白降解过程、形态发生和 主持人互动。RNA微核糖核酸病毒是最被理解和最容易获得的病毒之一。 所有生物学中的实验系统。自然感染心脏病毒，如脑心肌炎病毒 (EMCV)，在3天内几乎杀死小鼠大脑、胰腺和心脏中的每一个细胞。病毒就是这么做的 通过颠覆先天免疫陷阱和削弱 被感染细胞进行防御或触发警报的能力。内部的分子战场 感染细胞将病毒蛋白酶3CPro和两种独特的心脏病毒蛋白L和2A切割成两种酶，这些酶经过 为了它们特殊的抗细胞目的而进化，对抗完整的固有宿主防御。这个 结果几乎没有什么不同。在感染EMCV的2-3小时内，细胞内的mRNA转录就会停止，CAP- 依赖的mRNA翻译，抗病毒信号转导和活跃的蛋白质/RNA交换 胞核和细胞质。病毒大量复制，细胞在触发警报之前就死亡了。 在最终的分子水平上，这些蛋白质的活动引发了一连串的事件，从而引发或 预防一次疾病发作。这个项目的下一阶段将研究生物化学和分子 第一个结合和失活RAN GTP酶的病毒(或细胞)蛋白--EMCV L(Leader)的通路 循环，关键的，无处不在的监管系统，所有蛋白质和核酸进出 原子核。该项目还考察了L或L-然复合体的存在途径 激活一组特定的细胞激酶，并将它们重定向到核孔的磷酸化 蛋白质(NUP)。具体目的是：(1)对蒙古族L(领队)蛋白质的核磁共振结构进行解析。 与RAN GTP酶相互作用。(2)表征心脏病毒L的生物化学：RAN相互作用 在无细胞提取物中抑制RanGDP/GTP循环。(3)在细胞内鉴定L激活的宿主激酶， 这有助于废除核质运输步骤。(4)界定分子 L与其他病毒蛋白(2A和3CD)的相互作用，并确定其复制优势 心脏病毒，编码RAN和细胞蛋白和mRNA的独特和有效的抑制物 贩卖人口。这些目标直接建立在前一阶段开发的实验基础上 27年的计划。

The goals of this investigation are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family and to exploit the unique features of cardioviruses to examine fundamental molecular questions about picornavirus translation, proteolytic processing, morphogenesis and host interaction. The RNA picornaviruses are one of the best understood and most thoroughly accessible experimental systems in all of biology. Natural infections with cardioviruses, like encephalomyocarditis virus (EMCV), kill nearly every cell in the brain, pancreas and heart of a mouse, within 3 days. The virus does this with apparent impunity to cellular antiviral defenses by subverting innate immunity traps and crippling the capacity of an infected cell to mount a defense or trigger an alarm. The molecular battleground inside infected cells pits viral protease 3Cpro and two unique cardiovirus proteins, L and 2A, enzymes honed by evolution for their special anti-cellular purposes, against the complete array of innate host defenses. The outcome rarely varies. Within 2-3 hours of infection EMCV brings to a halt cellular mRNA transcription, cap- dependent mRNA translation, antiviral signal transduction, and active protein/RNA exchange between the nucleus and cytoplasm. The virus replicates with fecundity and the cell dies before it ever triggers an alarm. At the ultimate molecular level, the activities of these proteins instigate the cascade of events that set off or prevent an episode of disease. The next phase of this project will examine the biochemistry and molecular pathways of EMCV L (Leader), the first viral (or cellular) protein known to bind and inactivate Ran GTPase cycling, the crucial, ubiquitous regulatory system for all protein and nucleic acid trafficking into and out of the nucleus. The project also examines the pathways by which the presence of L, or L-Ran complexes activate a specific cohort of cellular kinases, and redirects them towards the phosphorylation of nuclear pore proteins (Nups). The specific aims are: (1) To resolve the NMR structure of Mengo L (Leader) protein as it interacts with Ran GTPase. (2) To characterize the biochemistry of cardiovirus L:Ran interactions which inhibit RanGDP/GTP cycling in cell-free extracts. (3) To identify within cells, the host kinases activated by L, which contribute to the abrogation of nucleocytoplasmic trafficking steps. (4) To define the molecular interactions of L with other viral proteins (2A and 3CD), and define the replication advantages to cardioviruses, for encoding a unique and potent inhibitor of Ran and of cellular protein and mRNA trafficking. These objectives build directly upon experimental foundations developed during the preceding 27 years of the program.