Herpesvirus protein kinases: Substrate recognition and pathway targeting.

描述（由申请人提供）：疱疹病毒是重要的人类病原体，可引起广泛的临床疾病，从相对良性的再活化感染（如唇疱疹）到更致命的癌症。年龄很小、老年人和免疫功能低下的人群，如移植患者和HIV-1感染者，更容易出现更严重的疾病结果。爱泼斯坦巴尔病毒（EBV）和卡波西肉瘤相关疱疹病毒（KSHV）与HIV-1阳性个体中的AIDS相关癌症和人巨细胞病毒（HCMV）与严重视网膜炎和肺炎相关。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危及生命的感染重新激活，HCMV导致移植排斥反应的发生率增加，EBV与移植后淋巴组织增生性疾病相关。 有有限数量的药物可用于治疗疱疹病毒感染。由这种有限的库引起的问题是耐药性病毒株的发展。因此，需要用于治疗疱疹病毒感染的新的治疗方法。人疱疹病毒各自编码丝氨酸/苏氨酸蛋白激酶。依赖于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以及它们的酶性质使得这些蛋白质成为抗病毒药物开发的有吸引力的靶标。我们还提出，确定关键的细胞蛋白或途径激活的病毒蛋白激酶将允许开发组合抗细胞蛋白加抗蛋白激酶治疗策略，这将限制耐药病毒变异体的发展。我们提出了两种方法，旨在获得基本的信息，这将有助于发展针对病毒蛋白激酶的抗疱疹病毒治疗。病毒蛋白激酶底物识别与细胞cdc 2/CDK 1激酶重叠，但延伸到cdc 2位点之外。扩展位点识别对于不同疱疹病毒蛋白激酶是独特的或共同的程度尚不清楚。将使用共同研究者开发的算法和本人实验室使用人蛋白质组阵列鉴定的644种EBV、HCMV、KSHV和HSV-1底物的数据库，采用生物信息学方法进行蛋白激酶位点鉴定。预测的基序将在体外磷酸化测定和转染和感染的细胞提取物中进行验证。我们最近确定TIP 60作为蛋白激酶的细胞底物，对疱疹病毒复制至关重要。使用EBV系统 作为模型，我们将通过在强力霉素诱导BGLF 4和对照激酶死亡的BGLF 4后询问Akata B细胞DNA的全基因组CpG甲基化来鉴定其表达受BGLF 4/TIP 60介导的染色质重塑调节的细胞基因。 将使用生物信息学分析挖掘筛选数据。将验证选定的靶标，并确定其敲除对病毒复制的影响。这些实验将揭示更多的细胞蛋白或网络，这些蛋白或网络可能成为抗病毒治疗策略的靶点。

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.