Human cytomegalovirus-induced alterations to cell-surface adhesion proteins

 描述(申请人提供)：人类巨细胞病毒(HCMV)感染超过60%的成年人口。它是导致出生缺陷的主要原因，也是免疫抑制人群中危及生命的机会性因素。先天性感染的发病率很高，可能导致永久性残疾。最近我们实验室的蛋白质组学分析显示，在人巨细胞病毒感染成纤维细胞后，许多细胞表面蛋白被动态调节，其中包括27个细胞表面黏附蛋白。两种上调的黏附蛋白，L1CAM和GPR56，已经被进一步测试，并被证明显著影响HCMV后代的产量。这些观察结果形成了支持这一应用的中心假设的基础：多个细胞黏附蛋白协同调节HCMV感染的结果。为了解决这一假设，我们将首先研究IgCAM家族成员L1CAM，它在广泛的人类肿瘤中升高，诱导辐射抵抗，并经常促进肿瘤的进展或转移。我们的初步研究表明，细胞表面的L1CAM在感染后升高，并支持在成纤维细胞中产生HCMV后代。感染后分泌的L1CAM胞外区(L1CAM-ECD，支持自分泌和旁分泌信号转导)和核L1CAM胞内区(L1CAM-ICD调节细胞基因表达)也显著增加。此外，L1CAM的敲除使感染细胞对拟放射药物敏感。L1CAM记录的一系列活动为详细研究其在HCMV复制和传播中的作用提供了令人信服的理由，即L1CAM可能在多个水平上影响HCMV的复制和传播：对HCMV后代的生产产生直接影响；通过感染细胞迁移对病毒传播的可能影响；通过L1CAM-ECD活性对感染细胞微环境的影响；以及感染细胞的辐射敏感性/DNA损伤反应。我们还将研究G蛋白偶联受体黏附家族的成员GPR56。我们对HCMV感染细胞蛋白质组的初步研究使用了成纤维细胞，这些成纤维细胞在体内的许多位置被感染，并作为参考，因为它们是研究HCMV的常见实验室宿主细胞。接下来，我们将描述上皮细胞的特征，以扩大我们对感染细胞表面蛋白质组的理解，并确定进一步的详细研究候选对象，如上面为L1CAM提出的那些研究。上皮细胞是巨细胞病毒致病的中心细胞，因为它们是在进入部位感染的，它们在唾液腺、乳腺和肾脏长期低水平复制，介导HCMV传播到新的宿主，它们是CMV视网膜炎的靶细胞。总而言之，这项研究将提供第一个广泛的基础，在巨细胞病毒感染过程中黏附蛋白的机制表征。这些研究的成功完成将有助于理解一个相对研究较少的巨细胞病毒生物学领域，即细胞黏附蛋白在病毒复制和传播中的作用。

 DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) infects over 60% of the adult population. It is a major cause of birth defects, and a life-threatening opportunistic agent in immunosuppressed people. Congenital infection has a significant prevalence and can cause permanent disabilities. Proteomic analysis in our lab has recently revealed that numerous cell-surface proteins are dynamically modulated following HCMV infection of fibroblasts, including 27 cell-surface adhesion proteins. Two upregulated adhesion proteins, L1CAM and GPR56, have been tested further and shown to significantly impact the yield of HCMV progeny. These observations form the foundation for the central hypothesis underlying this application: multiple cellular adhesion proteins cooperate to modulate the outcome of HCMV infection. To address this hypothesis, we will first study the IgCAM family member, L1CAM, which is elevated in a wide range of human tumors, inducing radioresistance and often enhancing tumor progression or metastasis. Our preliminary studies show that cell-surface L1CAM is elevated by infection and supports the production of HCMV progeny in fibroblasts. Secreted L1CAM extracellular domain (L1CAM-ECD, sponsors autocrine and paracrine signal transduction) and nuclear L1CAM intracellular domain (L1CAM-ICD modulates cellular gene expression) also are dramatically increased following infection. Further, knockdown of L1CAM sensitized infected cells to radiomimetic drugs. The remarkable range of activities documented for L1CAM provide a compelling rationale for detailed study of its role in HCMV replication and spread, i.e., L1CAM is likely to impact HCMV replication and spread at multiple levels: a direct effect on the production of HCMV progeny; and possible effects on viral spread via infected-cell migration, the infected-cell microenvironment via L1CAM-ECD activity, and the radiosensitivity/DNA-damage response of infected cells. We also will investigate GPR56, a member of the adhesion family of G protein-coupled receptors. Our initial studies of the HCMV-infected cell proteome used fibroblasts, which are infected at numerous sites within the body and serve as a reference, since they are the common laboratory host cell for HCMV studies. We will next characterize epithelial cells to expand our understanding of the infected cell-surface proteome and identify further candidates for detailed studies such as those proposed above for L1CAM. Epithelial cells are central to HCMV pathogenesis, because they are infected at the site of entry, they host long-term low-level replication in the salivary gland, breast and kidney to mediate HCMV spread to new hosts, and they are the target cell in CMV retinitis. Collectively, this study will provide he first broad-based, mechanistic characterization of adhesion proteins during HCMV infection. Successful completion of these studies should contribute importantly to the understanding of a relatively understudied area of HCMV biology, the role of cellular adhesion proteins in viral replication and dissemination.