Cardioviral Proteases and Comparative Genome Structure

描述（由申请人提供）：本研究的目的是探索和定义心脏病毒属与其他小核糖核酸病毒家族成员的关系，并利用心脏病毒的独特特征来研究有关小核糖核酸病毒翻译，蛋白水解加工，形态发生和宿主相互作用的基本分子问题。RNA小核糖核酸病毒是所有生物学中最容易理解和最彻底的实验系统之一。心脏病毒的自然感染，如脑心肌炎病毒（EMCV），可以在3天内杀死老鼠大脑、胰腺和心脏的几乎所有细胞。这种病毒通过破坏先天免疫陷阱，削弱受感染细胞进行防御或触发警报的能力，明显不受细胞抗病毒防御的影响。受感染细胞内的分子战场是病毒蛋白酶3Cpro和两种独特的心脏病毒蛋白L和2A，这两种酶经过进化，具有特殊的抗细胞作用，可以对抗宿主的一系列先天防御。结果很少变化。在感染后2-3小时内，EMCV使细胞mRNA转录、帽依赖性mRNA翻译、抗病毒信号转导以及细胞核和细胞质之间活跃的蛋白质/RNA交换停止。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系列引发或预防疾病发作的事件。该项目的下一阶段将研究EMCV L （Leader）的生物化学和分子途径，EMCV L是已知的第一个结合和灭活Ran GTPase循环的病毒（或细胞）蛋白，是所有蛋白质和核酸进出细胞核的关键、无处不在的调节系统。该项目还研究了L或L- ran复合物激活特定细胞激酶的途径，并将它们重新定向到核孔蛋白（Nups）的磷酸化。具体目的是：(1)解析Mengo L （Leader）蛋白与Ran GTPase相互作用时的NMR结构。(2)研究心脏病毒L:Ran相互作用抑制RanGDP/GTP循环的生物化学特征。(3)在细胞内识别L激活的宿主激酶，这些激酶有助于消除核细胞质运输步骤。(4)确定L与其他病毒蛋白（2A和3CD）的分子相互作用，并确定其对心脏病毒的复制优势，因为它编码了一种独特而有效的Ran和细胞蛋白和mRNA运输抑制剂。这些目标直接建立在项目前27年的实验基础上。公共卫生相关性：如果第一个被感染的细胞或宿主免疫系统能够有效地反击，很少会出现导致疾病的生产性病毒感染。在一场悄无声息的战争中，无意中触发细胞内或细胞外的免疫警报通常会给病毒带来灾难（和清除）。在感染的前2-3小时内产生的第一批病毒蛋白的作用是关闭基本的宿主反应系统。该项目研究了心脏病毒属中RNA小核糖核酸病毒如何发生这种情况的分子途径。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: It is rare to establish productive viral infections that lead to disease if the first infected cells or host immune system are able to fight back effectively. In a war won by stealth, an inadvertant triggering of intracellular or extracellular immunological alarms usually spells disaster (and clearance) for the virus. It's the job of the first viral proteins produced 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 picornaviruses in the cardiovirus genus.