Study of Bone Marrow Failure Caused by B19 Virus Infection

描述(申请人提供)：细小病毒B19(B19V)感染通过破坏支持病毒自主复制的骨髓常驻红系祖细胞(EPC)而导致人类骨髓衰竭。在由于高水平的红细胞破坏而对红细胞产生高需求的患者中(例如，镰状细胞病患者)，急性B19V感染可导致一过性再生障碍性危机。在免疫功能低下的患者中，持续的B19V感染可能表现为纯红细胞再生障碍性贫血。在胎儿中，B19V感染会导致胎儿积水，这是一种严重的贫血形式。B19V感染导致内皮祖细胞损伤，导致上述骨髓衰竭。感染B19V的EPC经历细胞死亡，具有明显的凋亡特征。这种细胞死亡主要是由感染过程中非结构性11 kDa蛋白(11 KDa)的高表达诱导的，并涉及由caspase-10触发的caspase级联反应。在这项拟议的研究中，我们的目标是，首先揭示caspase-10在11 kDa反应中介导的细胞凋亡的分子机制。为此，我们将确定在B19V感染诱导细胞凋亡过程中，外源性或内在哪一种凋亡途径激活了启动子caspase-10。由于凋亡和抗凋亡通路在红细胞生成和内皮祖细胞内稳态中都起着关键作用，我们建议鉴定与11 kDa相互作用的细胞蛋白，并在内皮祖细胞中转导凋亡信号。其次，最近建立了一个B19V培养系统，在低氧条件下模拟骨髓中人内皮祖细胞的天然B19V感染，我们将对小分子文库(可在堪萨斯大学HTS核心实验室获得；包含超过100,000个类似药物的小化合物)进行高通量筛选(HTS)，以寻找抗B19V感染的候选药物。B19V培养系统可以产生足够数量的病毒进行这种筛查，细胞死亡标记可以很容易地应用于评估B19V感染作为读数。我们将使用基于细胞的发光细胞毒性试验来筛选文库。由于B19V疫苗目前无法获得，而且免疫功能低下的患者将无法对疫苗产生反应，因此目前还没有可行的治疗方案来减少B19V感染引起的急性或持续B19V感染患者的骨髓衰竭。这项申请中提出的研究有望确定能够从人内皮祖细胞中消除B19V的药物开发的靶点，这最终将减少这些患者的骨髓衰竭。此外，我们对内皮祖细胞凋亡机制的分析有望揭示在某些疾病条件下可能参与红细胞生成或破坏红细胞生成的途径。 公共卫生相关性：人类细小病毒B19对人类是致病的，在一些严重的情况下会导致骨髓衰竭。对B19感染诱导的细胞死亡的研究将有助于我们从总体上了解B19导致骨髓衰竭的机制。因此，我们提出的工作将回答微小病毒B19感染的发病机制中的关键问题，并有可能确定用于治疗B19感染引起的骨髓衰竭的抗B19药物的候选药物。

DESCRIPTION (provided by applicant): Parvovirus B19 (B19V) infection causes human bone marrow failure by destroying the bone marrow resident erythroid progenitor cells (EPCs), which support autonomous replication of the virus. In patients with a high demand for erythrocyte production due to high levels of erythrocyte destruction (e.g., sickle-cell disease patients), acute B19V infection can cause transient aplastic crisis. In immunocompromised patients, persistent B19V infection may manifest as pure red-cell aplasia. In the fetus, B19V infection can cause hydrops fetalis, a severe form of anemia. B19V infection induces damage of EPCs, which results in the above bone marrow failures. B19V-infected EPCs undergo cell death with clear apoptotic features. This cell death is induced mainly by high expression of the non-structural 11kDa protein (11kDa) during infection, and involves a caspase cascade triggered by caspase-10. In the proposed research, we aim, firstly, to reveal the molecular mechanism underlying caspase-10- mediated apoptosis in response to 11kDa. To this end, we will determine which apoptotic pathway the extrinsic or the intrinsic activates the initiator caspase-10 during B19V infection-induced apoptosis. As both the apoptotic and anti-apoptotic pathways play key roles in erythropoiesis and the homeostasis of EPCs, we propose to identify the cellular proteins that interact 11kDa and transduce the apoptotic signal in EPCs. Secondly, having recently established a B19V culture system that mimics native B19V infection of human EPCs in bone marrow under hypoxic conditions, we will carry out high throughput screening (HTS) of a library of small molecules (available at the University of Kansas HTS Core Laboratory; contains over 100,000 druglike small compounds) for anti-B19V infection drug candidates. The B19V culture system can generate virus in sufficient quantity for such screening, and the cell death marker can be readily applied to assess B19V infection as a readout. We will use a cell-based luminescent cytotoxicity assay to screen the library. Because B19V vaccine is currently unavailable, and immunocompromised patients would be unable to respond to a vaccine, there are currently no viable treatment options for diminishing the bone marrow failure caused by B19V infection in patients with acute or persistent B19V infection. The study proposed in this application is expected to identify targets for the development of drugs capable of eliminating B19V from human EPCs, and this will ultimately lead to diminished bone marrow failures in these patients. In addition, our analysis of the mechanism underlying apoptosis of EPCs is expected to reveal pathways that may be involved in erythropoiesis or the disruption of erythropoiesis under certain disease conditions. PUBLIC HEALTH RELEVANCE: Human parvovirus B19 is pathogenic to humans, causes bone marrow failure in some severe circumstances. Study of B19 infection-induced cell death will help us to understand the mechanism of B19 caused bone marrow failure in general. Our proposed work will thus answer critical questions in the pathogenesis of parvovirus B19 infection, and have the potential to identify candidates of anti-B19 drugs for treating B19 infection-caused bone marrow failure.