UBIQUITIN AND CELLULAR FACTORS IN CORONAVIRUS ASSEMBLY

描述(申请人提供)：冠状病毒可引起禽类和动物的呼吸道和胃肠道疾病。这些RNA病毒在基因上是可变的，可以迅速进化到感染人类，有时会导致严重的急性呼吸道疾病。冠状病毒通过一种涉及细胞内病毒形成和可能向外部环境传播的新途径的策略，在它们的组装和分泌方面与细胞分开。对这些晚期感染事件的进一步了解将确定治疗干预的策略。我们已经发现，小鼠肝炎冠状病毒原型在组装和/或分泌阶段被极低浓度的无毒蛋白酶体抑制剂抑制。我们假设抑制机制涉及细胞泛素耗尽，这是蛋白酶体抑制剂的已知效应，因为我们发现病毒分泌中心的病毒E蛋白泛素存在于两个赖氨酸残基上。我们的目的是确定致病性人类冠状病毒是否对蛋白酶体抑制剂类似地超敏，然后解决E蛋白的泛素结合是否是冠状病毒形态发生或清除细胞的中心。我们的实验将专门评估抑制物和泛素的作用中心是否处于病毒分泌阶段，并将解决泛素修饰引导充满病毒的细胞器运输到细胞表面的新假设，在那里病毒货物被释放。我们的发现将揭示囊泡形成和细胞器运输的新的细胞生物学特征，并将告诉我们在感染后期阻止冠状病毒的潜力。公共卫生相关性：冠状病毒会导致人类和家养动物的呼吸道疾病，在严重急性呼吸综合征冠状病毒的病例中，这些症状可能危及生命。冠状病毒以一种新的方式形成，它组装成细胞器小泡，然后当小泡融合到质膜上时从细胞中排出。由于对这一过程如何发生知之甚少，该提案旨在揭开冠状病毒从细胞中组装和分泌的机制，从而揭示干预这一关键感染阶段的目标。

DESCRIPTION (provided by applicant): Coronaviruses cause respiratory and gastrointestinal diseases in birds and animals. These RNA viruses are genetically variable and can rapidly evolve to infect humans, sometimes causing severe acute respiratory disease. The coronaviruses are set apart in their assembly and secretion from cells by a strategy involving intracellular virus formation and perhaps novel trafficking routes to the outside environment. Further understanding of these late infection events will identify strategies for therapeutic intervention. We have discovered that a prototype mouse hepatitis coronavirus is inhibited at the assembly and / or secretion stages by very low nontoxic concentrations of a proteasome inhibitor. We hypothesize that the inhibitory mechanism involves ubiquitin depletion from cells, a known effect of proteasome inhibitors, because we discovered that the viral E proteins that are central to virus secretion are ubiquitinated on two lysine residues. Our aims are to determine whether pathogenic human coronaviruses are similarly hypersensitive to proteasome inhibitors and then address whether the ubiquitin conjugation of E proteins is central to coronavirus morphogenesis or expulsion out of cells. Our experiments will specifically evaluate whether the locus of inhibitor and ubiquitin action are at the virus secretion stages and will address the novel hypothesis that ubiquitin modifications direct the trafficking of virus-filled organelles to cell surfaces where the virus cargo is liberated. Our findings will reveal novel cell biological features of vesicle formation and organelle transport and will also inform us about the potential to thwart coronaviruses at late infection stages. PUBLIC HEALTH RELEVANCE: Coronaviruses cause respiratory diseases in humans and domesticated animals and in cases of severe acute respiratory syndrome coronavirus the symptoms can be life threatening. Coronaviruses form in a novel fashion by assembling into organellar vesicles and then expelling from cells when the vesicles fuse to the plasma membrane. As little is known about how this process occurs, the proposal aims to unravel mechanisms of coronavirus assembly and secretion from cells and thereby uncover targets for intervention with this essential infection stage.