Herpesvirus protein kinases: Substrate recognition and pathway targeting.

描述(申请人提供)：疱疹病毒是一种重要的人类病原体，可导致广泛的临床疾病，从相对良性的复活感染，如唇疱疹到更致命的癌症。非常年轻的人、老年人和免疫功能低下的人群，如移植患者和感染艾滋病毒-1的人，容易出现更严重的疾病后果。在HIV-1感染者中，Epstein Barr病毒(EBV)和Kaposi肉瘤相关疱疹病毒(KSHV)与艾滋病相关癌症和人类巨细胞病毒(HCMV)合并严重的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作为播散性威胁生命的感染重新激活，HCMV导致移植物排斥反应的发生率增加，EBV与移植后淋巴增生性疾病有关。可用于治疗疱疹病毒感染的药物数量有限。这一有限的保存库带来的一个问题是抗药性病毒株的开发。因此，需要治疗疱疹病毒感染的新的治疗方法。人类疱疹病毒每一种都编码一种丝氨酸/苏氨酸蛋白激酶。依赖这些蛋白激酶进行有效的病毒复制和传播，它们在疱疹病毒家族中的保守性和它们的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点。我们还建议，识别关键的细胞蛋白或由病毒蛋白激酶激活的通路将允许开发抗细胞蛋白+抗蛋白激酶的组合治疗策略，从而限制耐药病毒变体的发展。我们提出了两种旨在获得基本信息的方法，以促进针对病毒蛋白激酶的抗疱疹病毒治疗的发展。病毒蛋白激酶底物识别与细胞cdc2/cdk1激酶重叠，但延伸到cdc2以外。扩展的位点识别对于不同的疱疹病毒蛋白激酶是独特的还是共同的，目前尚不清楚。利用联合调查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底物的数据库，将采用生物信息学的方法来鉴定蛋白激酶位点。预测的基序将在体外磷酸化试验中以及在转基因和感染细胞提取液中得到验证。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，我们将通过询问多西环素诱导BGLF4和对照激酶DEAD BGLF4后Akata B细胞DNA全基因组CpG甲基化来鉴定其表达受BGLF4/Tip60介导的染色质重塑的细胞基因。屏幕上的数据将使用生物信息学分析进行挖掘。将对选定的目标进行验证，并确定其击倒对病毒复制的影响。这些实验将发现更多的细胞蛋白质或网络，可能成为抗病毒治疗策略的目标。 与公共卫生相关：疱疹病毒可引起从唇疱疹到出生缺陷和癌症等多种人类疾病。目前只有一类药物可用于治疗疱疹病毒感染。这项申请建议获得有关疱疹病毒编码的蛋白激酶酶功能的信息，这些信息将有助于开发针对这些蛋白的新的抗病毒治疗方法。

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. PUBLIC HEALTH RELEVANCE: Herpesviruses cause a wide range of human diseases ranging from cold sores to birth defects and cancers. There is currently only one class of drugs available to treat herpesvirus infections. This application proposes to obtain information on the functioning of the herpesvirus encoded protein kinase enzymes that will assist in the development of new anti-viral treatment approaches targeting these proteins.