UBIQUITIN AND CELLULAR FACTORS IN CORONAVIRUS ASSEMBLY

描述（由申请人提供）：冠状病毒引起鸟类和动物的呼吸道和胃肠道疾病。这些RNA病毒在遗传上是可变的，可以迅速进化感染人类，有时会引起严重的急性呼吸道疾病。冠状病毒在细胞的组装和分泌过程中，通过一种涉及细胞内病毒形成和可能到外部环境的新运输途径的策略而与众不同。进一步了解这些晚期感染事件将确定治疗干预的策略。我们已经发现，一种原型小鼠肝炎冠状病毒在组装和/或分泌阶段被非常低的无毒蛋白酶体抑制剂抑制。我们假设抑制机制涉及细胞中泛素的消耗，这是蛋白酶体抑制剂的已知效应，因为我们发现病毒分泌的中心病毒E蛋白在两个赖氨酸残基上泛素化。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同样过敏，然后确定E蛋白的泛素结合是否对冠状病毒形态发生或排出细胞至关重要。我们的实验将专门评估抑制剂和泛素作用位点是否在病毒分泌阶段，并将解决泛素修饰指导满载病毒的细胞器运输到释放病毒货物的细胞表面的新假设。我们的发现将揭示囊泡形成和细胞器运输的新细胞生物学特征，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：冠状病毒在人类和家畜中引起呼吸道疾病，在严重急性呼吸综合征冠状病毒病例中，症状可能危及生命。冠状病毒以一种新颖的方式形成，通过组装成细胞器囊泡，然后当囊泡与质膜融合时从细胞中排出。由于对这一过程的发生机制知之甚少，该提案旨在揭示冠状病毒组装和细胞分泌的机制，从而揭示干预这一重要感染阶段的目标。

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.