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）：疱疹病毒是显着的人类病原体，导致广泛的临床疾病，从相对良性的重新激活感染（例如冷疮）到更致命的癌症。非常年轻的老年人和免疫功能低下的人群，例如移植患者和HIV-1感染患者，易受更严重的疾病结局的影响。爱泼斯坦Barr病毒（EBV）和Kaposi的肉瘤相关疱疹病毒（KSHV）与与HIV-1阳性个体中严重的视网膜炎和肺炎有关，与艾滋病相关的癌症和人类巨细胞病毒（HCMV）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以随着传播的威胁生命的感染而重新激活，HCMV会导致移植物排斥的发生率增加，而EBV与移植后植物淋巴淋巴结疾病有关。有限的药物可用于治疗疱疹病毒感染。由于这种有限的曲目引起的一个问题是耐药性病毒菌株的发展。因此，需要采用新的治疗疱疹病毒感染的治疗方法。人疱疹病毒每个编码丝氨酸/苏氨酸蛋白激酶。对这些蛋白激酶的依赖性用于有效的病毒复制和扩散，它们在疱疹病毒家族中的保护及其酶促性质使这些蛋白质具有吸引力的抗病毒药物开发靶标。我们还建议，鉴定由病毒蛋白激酶激活的关键细胞蛋白或途径将允许发展组合抗细胞蛋白以及抗蛋白激酶治疗策略，从而限制耐药性病毒变体的发展。我们提出了两种旨在获取基本信息的方法，这些信息将促进针对病毒蛋白激酶的抗赫普病毒治疗的发展。病毒蛋白激酶底物识别与细胞CDC2/CDK1激酶的识别重叠，但延伸超出Cdc2位点。尚不清楚扩展位点识别的程度是不同疱疹病毒蛋白激酶的独特或共同的程度。将使用由共同研究器开发的算法和644 EBV，HCMV，KSHV和HSV-1底物在我的实验室中使用人类蛋白质组学阵列确定的644 EBV，HCMV，KSHV和HSV-1底物的数据库进行生物信息学方法。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进行验证。我们最近将TIP60确定为蛋白激酶的细胞底物，这对于疱疹病毒复制至关重要。使用EBV系统作为该模型，我们将通过对BGLF4和控制激酶死激素酶死亡的BGLF4的基因组宽CPG甲基化来鉴定其表达受到BGLF4/TIP60介导的染色质重塑调节的细胞基因。屏幕上的数据将使用生物信息学分析挖掘。选定的靶标将得到验证，并确定其敲低对病毒复制的影响。这些实验将发现可能是抗病毒治疗策略的靶标的其他细胞蛋白或网络。