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Herpesvirus protein kinases: Substrate recognition and pathway targeting.

疱疹病毒蛋白激酶：底物识别和通路靶向。

基本信息

批准号：
8546298
负责人：
S DIANE HAYWARD
金额：
$ 19.04万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-17 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8546298
关键词：
AIDS related cancer Algorithms B-Lymphocytes Benign Binding Binding Proteins Bioinformatics Biological Assay CDC2 Protein Kinase Cell Extracts Cell Line Cell physiology Cells Chromatin Clinical Complex Congenital Abnormality Consensus Cyclin-Dependent Kinases Cytomegalovirus DNA DNA Damage DNA Methylation Data Data Set Databases Dependence Development Disease Disease Outcome Distal Doxycycline Drug Targeting Elderly Environment Enzymes Epstein-Barr virus BGLF4 protein Family Gene Expression Gene Expression Regulation Genes Graft Rejection HIV-1 HTATIP gene Herpes Labialis Herpesviridae Herpesviridae Infections Herpesvirus 1 Herpesvirus Type 3 Heterochromatin Histone Acetylation Histones Human Human Herpesvirus 4 Human Herpesvirus 8 Immunocompromised Host In Vitro Incidence Individual Infection Laboratories Life Lymphoproliferative Disorders Malignant Neoplasms Mediating Methylation Mining Modeling Monitor Motion Mutation Nature Nucleic Acid Regulatory Sequences Orthologous Gene Pathway interactions Pharmaceutical Preparations Phosphorylation Phosphotransferases Pneumonia Population Post-Translational Protein Processing Protein Array Protein Kinase Protein Kinase Inhibitors Protein-Serine-Threonine Kinases Proteins Proteomics Regulation Research Personnel Retinitis Role Screening Result Simplexvirus Site System Therapeutic Therapeutic Intervention Transplant Recipients Transplantation Ubiquitin Like Proteins Variant Viral Viral Proteins Virus Virus Replication chromatin remodeling combinatorial design drug development drug resistant virus gammaherpesvirus genome-wide histone acetyltransferase human disease knock-down novel therapeutic intervention pathogen preference protein kinase inhibitor research study response

项目摘要

DESCRIPTION (provided by applicant): Herpes viruses are significant human pathogens causing a wide range of clinical diseases ranging from relatively benign reactivated infections such as cold sores to more deadly cancers. The very young, the elderly and immunocompromised populations, such as transplant patients and those with HIV-1 infections, are susceptible to more serious disease outcomes. Epstein Barr Virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV) are associated with AIDS related cancers and human cytomegalovirus (HCMV) with severe retinitis and pneumonitis in HIV-1 positive individuals. In transplant patients, herpes simplex virus (HSV) and varicella zoster virus can reactivate as disseminated life-threatening infections, HCMV causes an increased incidence of graft rejection and EBV is associated with post-transplant lymphoproliferative disease. There are a limited number of drugs available to treat herpesvirus infections. A problem arising from this limited repertoire is the development of drug resistant virus strains. New therapeutic approaches for treating herpesvirus infections are therefore needed. The human herpesviruses each encode a serine/threonine protein kinase. The dependence on these protein kinases for efficient virus replication and spread, their conservation across the herpesvirus family and their enzymatic nature make these proteins attractive targets for anti-viral drug development. We also propose that identifying key cellular proteins or pathways activated by the virus protein kinases would permit the development of combinatorial anti-cell protein plus anti-protein kinase therapeutic strategies that would limit the development of drug resistant virus variants. We present two approaches designed to obtain basic information that would facilitate the development of anti-herpesvirus treatments targeted at the viral protein kinases. Viral protein kinase substrate recognition overlaps with that of cellular cdc2/CDK1 kinase but is extended beyond cdc2 sites. The degree to which the extended site recognition is unique to, or common to, the different herpesvirus protein kinases is not known. A bioinformatic approach to protein kinase site identification will be undertaken using an algorithm developed by the Co-Investigator and a database of 644 EBV, HCMV, KSHV and HSV-1 substrates identified in my laboratory using human proteomic arrays. The predicted motifs will be validated in in vitro phosphorylation assays and in transfected and infected cell extracts. We recently identified TIP60 as a cell substrate of the protein kinases that is critical for herpesvirus replication. Using the EBV system as the model, we will identify cell genes whose expression is regulated by BGLF4/TIP60 mediated chromatin remodeling by interrogating genome wide CpG methylation of Akata B cell DNA after doxycycline induction of BGLF4 and control kinase dead BGLF4. Data from the screen will be mined using bioinformatic analyses. Selected targets will be validated and the effect of their knockdown on virus replication determined. These experiments will uncover additional cell proteins or networks that could be targets for anti-viral therapeutic strategies.

描述（由申请人提供）：疱疹病毒是重要的人类病原体，可引起多种临床疾病，从相对良性的再激活感染（例如唇疱疹）到更致命的癌症。幼儿、老年人和免疫功能低下的人群，例如移植患者和 HIV-1 感染者，容易出现更严重的疾病结果。 EB 病毒 (EBV) 和卡波西肉瘤相关疱疹病毒 (KSHV) 与艾滋病相关癌症有关，而人类巨细胞病毒 (HCMV) 则与 HIV-1 阳性个体的严重视网膜炎和肺炎有关。在移植患者中，单纯疱疹病毒 (HSV) 和水痘带状疱疹病毒可重新激活，成为危及生命的播散性感染，HCMV 导致移植物排斥发生率增加，EBV 与移植后淋巴细胞增殖性疾病相关。可用于治疗疱疹病毒感染的药物数量有限。这种有限的病毒库产生的一个问题是耐药病毒株的发展。因此需要治疗疱疹病毒感染的新治疗方法。人类疱疹病毒各自编码丝氨酸/苏氨酸蛋白激酶。病毒有效复制和传播依赖于这些蛋白激酶，它们在疱疹病毒家族中的保守性及其酶性质使这些蛋白质成为抗病毒药物开发的有吸引力的靶标。我们还建议，识别病毒蛋白激酶激活的关键细胞蛋白或途径将允许开发抗细胞蛋白加抗蛋白激酶组合治疗策略，从而限制耐药病毒变体的发展。我们提出了两种旨在获取基本信息的方法，这些信息将有助于开发针对病毒蛋白激酶的抗疱疹病毒治疗方法。病毒蛋白激酶底物识别与细胞 cdc2/CDK1 激酶的底物识别重叠，但扩展到 cdc2 位点之外。不同疱疹病毒蛋白激酶的扩展位点识别独特或共有的程度尚不清楚。将使用共同研究者开发的算法和我的实验室使用人类蛋白质组阵列鉴定的 644 个 EBV、HCMV、KSHV 和 HSV-1 底物数据库，采用生物信息学方法识别蛋白激酶位点。预测的基序将在体外磷酸化测定以及转染和感染的细胞提取物中得到验证。我们最近发现 TIP60 是对疱疹病毒复制至关重要的蛋白激酶的细胞底物。使用 EBV 系统作为模型，我们将通过在强力霉素诱导 BGLF4 和对照激酶死亡 BGLF4 后询问 Akata B 细胞 DNA 的全基因组 CpG 甲基化来鉴定其表达受 BGLF4/TIP60 介导的染色质重塑调节的细胞基因。来自屏幕的数据将使用生物信息学分析进行挖掘。选定的目标将得到验证，并确定其敲低对病毒复制的影响。这些实验将揭示其他细胞蛋白或网络，它们可能成为抗病毒治疗策略的目标。