Human Cytomegalovirus Terminase: Insights into structure-function relationships

人巨细胞病毒（Human cytomegalovirus，HCMV）属于疱疹病毒属，具有宿主范围窄的特点. HCMV血清阳性率在世界范围内超过50%，并且HCMV感染在免疫功能低下的患者（例如，移植受体和AIDS患者）中导致严重的炎性疾病。目前可用的药物效果有限，并导致耐药性。HCMV成熟的关键步骤是DNA复制和包装成衣壳，这需要将串联的DNA切割成单位长度的基因组。该过程在哺乳动物细胞中未发现，因此是抗HCMV治疗的有希望的靶点。我们已经证明，HCMV末端酶，由两个亚基，pUL 56和pUL 89，参与切割过程。最近，我们在体外确定了两个潜在的DNA结合结构域的C-末端部分的终止酶亚基pUL 56和一个在N-末端部分的亚基pUL 89。该功能的要求是末端酶亚基的相互作用。在这个项目中，这些DNA结合结构域以及pUL 89的相互作用结构域将通过产生不同的HCMV缺失突变体来验证。突变病毒将表征其生长动力学、病毒颗粒形成及其DNA结合能力。拟议的工作将导致一个一致的HCMV DNA复制模型，并将是重要的高度特异性的抗病毒药物靶向的末端酶亚基的基本结构域的发展。

Human cytomegalovirus (HCMV) belongs to the ß-herpesviruses and is characterized by its narrow host range. HCMV seroprevalence is more than 50% worldwide, and HCMV infection leads to serious inflammatory disease in immunocompromised patients, e.g. transplant recipients and AIDS patients. Currently available drugs have limited effectiveness and lead to resistance. Key steps in HCMV maturation are DNA replication and packaging into capsids, which require cleavage of concatenated DNA into unit-length genomes. This process is not found in mammalian cells and therefore a promising target for anti-HCMV therapy. We have demonstrated that the HCMV terminase, consisting of two subunits, pUL56 and pUL89, is involved in the cleavage process. Recently we identified in vitro two potential DNA-binding domains in the C-terminal part of terminase subunit pUL56 and one in the N-terminal part of subunit pUL89. A requirement for the function is the interaction of the terminase subunits. In this project, these DNA-binding domains as well as the interaction domain of pUL89 will be validated by the generation of different HCMV deletion mutants. The mutant viruses will be characterized concerning their growth kinetics, viral particle formation and their DNA binding ability. The proposed work will lead to a consistent model of HCMV DNA replication and will be important for the development of highly specific antivirals targeting the essential domains of the terminase subunits.