Receptors and Signals in Coxsackievirus Pathogenesis

描述（由申请人提供）：柯萨奇B型病毒（CBV）与两种受体相互作用引发感染。所有CBV通过附着柯萨奇病毒和腺病毒受体（CAR）引发感染；许多CBV分离株还结合第二种受体，人类衰变加速因子（DAF）。与其他肠道病毒一样，CBV在人类中通过粪-口途径传播，并通过穿过肠黏膜引发感染；然而，CAR并不在肠腔内极化上皮细胞的顶端表面表达。在这项资助的第一个资助期，我们发现DAF对于极化细胞的感染是必不可少的。DAF允许病毒附着在细胞表面，但更重要的是，它诱导了病毒进入细胞所需的多种细胞内信号。我们发现额外的病毒诱导信号通过激活钙依赖性蛋白酶（钙蛋白酶）来启动非凋亡细胞死亡。基于这些观察结果，我们提出了一系列新的实验，重点关注病毒与DAF的相互作用，DAF在肠内途径感染中的作用，以及钙介导的死亡信号的机制和重要性。1. 我们将定义病毒表面允许附着DAF的位点，以及DAF分子上负责与病毒相互作用的位点，使用位点定向诱变来测试最近从冷冻电镜实验中获得的病毒-DAF复合物模型。2. 我们将利用在肠上皮上表达人DAF的转基因小鼠，来确定上皮细胞表面DAF的表达是否对肠内途径的体内感染有重要作用。3. 我们将确定导致培养肠上皮坏死细胞死亡的信号通路，确定calpain激活是否有助于感染动物心肌炎和胰腺炎的发病机制，并测试calpain抑制剂是否降低疾病的严重程度。

DESCRIPTION (provided by applicant): Coxsackie B viruses (CBV) interact with two receptors to initiate infection. All CBV initiate infection by attaching to the coxsackievirus and adenovirus receptor (CAR); many CBV isolates also bind to a second receptor, human decay accelerating factor (DAF). CBV, like other enteroviruses, are transmitted by the fecal-oral route in humans, and initiate infection by crossing the intestinal mucosa; however, CAR is not expressed on the apical surface of the polarized epithelial cells that line the intestinal lumen. In the first funding period of this grant, we have found that DAF is essential for infection of polarized cells. DAF permits virus to attach to the cell surface, but more importantly, it induces multiple intracellular signals that are required for virus uptake into the cell. We have found that additional virus-induced signals initiate non-apoptotic cell death by activating calcium-dependent proteases (calpains). Based on these observations, we propose a new series of experiments, focused on virus interaction with DAF, the role of DAF in infection by the enteral route, and the mechanisms and importance of calcium-mediated death signals. 1. We will define the site on the virus surface that permits attachment to DAF, and the site on the DAF molecule responsible for interaction with virus, using site-directed mutagenesis to test a model of the virus-DAF complex recently derived from cryo-em experiments. 2. We will determine whether DAF expression on the epithelial cell surface is important for in vivo infection by the enteral route, using transgenic mice expressing human DAF on intestinal epithelium. 3. We will define the signaling pathway that leads to necrotic cell death in cultured intestinal epithelium, determine whether calpain activation contributes to the pathogenesis of myocarditis and pancreatitis in infected animals, and test whether calpain inhibitors reduce the severity of disease. Relevance: Enteroviruses are the major cause of viral meningitis, and the second major cause of myocarditis in the US. Understanding how virus interacts with its intestinal receptor as it initiates infection, and understanding the mechanisms of virus-induced cell death, may provide new targets for therapeutic intervention.