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成员的关系。 微小核糖核酸病毒家族，并利用心脏病毒的独特特征来研究基本的分子问题 关于微小核糖核酸病毒的翻译、蛋白降解处理、形态发生和宿主相互作用。RNA小核糖核酸病毒是 这是所有生物学中最被理解和最彻底的实验系统之一。自然感染 心脏病毒，如脑心肌炎病毒(EMCV)，几乎杀死大脑、胰腺和心脏中的每一个细胞 老鼠，在3天内。这种病毒通过颠覆天生的细胞抗病毒防御系统而不受惩罚。 免疫陷阱和削弱感染细胞的能力--发动防御或触发警报。分子 在受感染的细胞内有病毒蛋白酶3CPro和两种独特的心脏病毒蛋白L和2A酶 经过进化磨练，以达到特殊的抗细胞目的，对抗完整的固有宿主防御系统。这个 结果几乎没有什么不同。在感染后2-3小时内，EMCV会导致细胞内依赖帽子的mRNA转录停止 信使核糖核酸的翻译，抗病毒信号转导，以及核质之间活跃的蛋白质/RNA交换。 病毒大量复制，细胞在触发警报之前就死亡了。在终极分子水平上， 这些蛋白质的活动引发了一连串的事件，从而引发或预防了疾病的发作。下一阶段 将研究第一个病毒(或细胞)L(领头人)的生化和分子途径 已知的结合和失活RAN GTP酶循环的蛋白质，是所有蛋白质和 核酸进出原子核的运输。该项目还延长了对EMCV 2A的研究，EMCV 2A是一种可以 颠覆核仁并将正常的核糖体转换为阻止宿主mRNAs翻译的配置，并且它 继续检查3CPro，一种其前体具有远远超出正常水平的抗宿主倾向的酶 病毒多蛋白的加工。这些目标直接建立在 在这项计划的26年前。具体目标是：(1)解析蒙果·L(领队)的核磁共振结构 蛋白质与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)在细胞内识别核质转运步骤 被心脏病毒L蛋白消灭。(4)明确心脏病毒的分子优势，编码有效的抑制物 对Ran来说。(5)阐明心脏病毒L、2A和3C在Pol2转录阻断中的作用。 被感染的细胞。 与公共卫生的相关性：如果首次感染病毒，很少能建立导致疾病的生产性病毒感染 宿主细胞或宿主免疫系统能够有效地反击。在一场秘密取胜的战争中，无意中引发了 细胞内或细胞外的免疫警报通常意味着病毒的灾难(和清除)。这是我们的工作 首先，病毒蛋白在感染的头2-3小时内产生，关闭必要的宿主反应系统。这个项目 研究了心脏病毒属中的RNA微管病毒是如何发生这种情况的分子途径。

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 extends studies on EMCV 2A, a protein that subverts nucleoli and converts normal ribosomes into configurations which prevent translation of host mRNAs, and it continues examinations of 3Cpro, an enzyme whose precursors have anti-host proclivities that go far beyond normal processing of the viral polyprotein. These objectives build directly upon experimental foundations developed during the preceding 26 years of the program. The specific aims of 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 nucleocytoplasmic trafficking steps abrogated by cardiovirus L protein. (4) To define molecular advantages to cardioviruses, for encoding a potent inhibitor of Ran. (5) To clarify the respective roles of cardioviral L, 2A and 3C, in pol-2 transcriptional shutoff within the nuclei of infected cells. Relevance to Public Health: It is rare to establish productive viral infections that lead to disease if the first infected host cells or host immune system are able to fight back effectively. In a war won by stealth, an inadvertent triggering of intracellular or extracellular immunological alarms usually spells disaster (and clearance) for the virus. It's the job of the first viral proteins produces in the first 2-3 hrs of infection, to shutoff essential host response systems. This project examines the molecular pathways for how this happens with RNA picornavi ruses in the cardiovirus genus.