Cardioviral Proteases and Comparative Genome Structure

描述(由申请人提供)：本研究的目标是探索和确定心脏病毒属与微小核糖核酸病毒家族其他成员的关系，并利用心脏病毒的独特特征来研究关于微小核糖核酸病毒翻译、蛋白分解处理、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被理解和最彻底的实验系统之一。自然感染的心脏病毒，如脑心肌炎病毒(EMCV)，在3天内几乎杀死小鼠大脑、胰腺和心脏的每一个细胞。这种病毒通过颠覆先天免疫陷阱，削弱受感染细胞进行防御或触发警报的能力，从而实现了对细胞抗病毒防御的明显不受惩罚。在感染细胞内的分子战场上，病毒蛋白酶3CPro和两种独特的心脏病毒蛋白L和2A是为了特殊的抗细胞目的而进化而成的酶，与完整的天然宿主防御系统相对抗。结果几乎没有什么不同。在感染后的2-3小时内，EMCV使细胞的mRNA转录、帽依赖的mRNA翻译、抗病毒信号转导以及胞核和胞浆之间活跃的蛋白质/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连串的事件，从而引发或预防了疾病的发作。该项目的下一阶段将研究EMCV L(Leader)的生化和分子途径，它是已知的第一种结合和失活RAN GTP酶循环的病毒(或细胞)蛋白，RAN GTP酶循环是所有蛋白质和核酸进出细胞核的关键、普遍存在的调节系统。该项目还研究了L或L-RAN复合体的存在激活特定一组细胞激酶的途径，并将它们重新定向到核孔蛋白(NUP)的磷酸化。具体目的是：(1)解析蒙果L(领队)蛋白与RAN GTP酶相互作用的核磁共振结构。(2)研究心脏病毒L的生化特征：在无细胞提取液中RAN相互作用抑制RanGDP/GTP循环。(3)在细胞内鉴定L激活的、有助于消除核质转运步骤的宿主蛋白。(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，以编码一种独特而有效的RAN和细胞蛋白和信使核糖核酸转运的抑制因子。这些目标直接建立在该计划前27年发展的实验基础上。与公共卫生相关：如果最初被感染的细胞或宿主免疫系统能够有效地反击，则很少能建立导致疾病的生产性病毒感染。在一场秘密取胜的战争中，不经意间触发的细胞内或细胞外免疫警报通常意味着病毒的灾难(和清除)。这是在感染的前2-3小时内产生的第一批病毒蛋白的工作，关闭了必要的宿主反应系统。这个项目研究了心脏病毒属中的RNA小核糖核酸病毒是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.