Characterizing the function of pUL33 of HSV-1 in viral DNA cleavage and packaging

描述(申请人提供)：单纯疱疹病毒(HSV)是一种重要的人类病原体，感染全球65%-90%的人口，主要导致口腔(HSV-1)或生殖器(HSV-2)黏膜皮肤溃疡。更严重的感染可能导致危及生命的脑炎，而单纯疱疹病毒感染引起的角膜炎是美国失明的主要原因。单纯疱疹病毒2型是全世界最流行的性传播疾病之一；它也是艾滋病毒传播的一个重要辅助因素。几种抗病毒药物已被批准用于治疗单纯疱疹病毒感染，但它们的疗效仅限于潜伏期确定之前的一段时间，并可能因出现耐药性而受到影响。因此，研究新的药物靶点和治疗策略非常重要。病毒DNA的裂解和包装成预先组装的衣壳是疱疹病毒复制生命周期中的关键步骤；理解这一过程具有重要的实际意义，因为它为开发新的抗疱疹病毒策略提供了丰富的靶点。这种DNA切割和包装反应是由终止酶介导的，终止酶是HSV中由pUL15、pUL28和pUL33组成的病毒编码酶。所有的末端酶亚基都是DNA包装所必需的，但个别亚基的作用尚不清楚。到目前为止，还没有发现与已知位点结合的病毒蛋白。 是启动DNA裂解/包装反应所必需的。我们最近在纯化和鉴定pUL33方面取得了重大进展。在初步数据中，我们显示pUL33与PAC2序列结合，并推测这有助于启动DNA切割/包装过程。利用我们实验室发展的方法，我们建议(I)表征pUL33的DNA结合活性，(Ii)绘制pUL33的功能结构域，以及(Iii)测试DNA结合活性是否在感染细胞中的DNA切割中起重要作用。这些研究将直接解决该领域的一个重要问题：终止酶如何识别病毒基因组中的包装信号，以启动DNA切割/包装过程。此外，对DNA切割/包装机制的研究可能会导致新的抗疱疹病毒策略的开发。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a significant human pathogen infecting 65%-90% of the population worldwide and primarily causing oral (HSV-1) or genital (HSV-2) mucocutaneous ulcers. More serious infections can result in life-threatening encephalitis, and keratitis from HSV infection is the leading cause of blindness in the United States. HSV-2 is one of the most prevalent sexually transmitted diseases worldwide; it is also a significant co-factor for HIV spread. Several antivirals are approved for the treatment of HSV infections, but their efficacy is limited to the period before latency is established and can be compromised by the emergence of drug resistance. It is, therefore, important to investigate new drug targets and treatment strategies. The cleavage and packaging of the viral DNA into preassembled capsids is a critical step in the life cycle of herpes virus replication; understandin this process has an important practical implication because it provides a rich target for developing novel anti-herpes virus strategies. This DNA cleavage and packaging reaction is mediated by terminase, a virally-encoded enzyme composed of pUL15, pUL28 and pUL33 in HSV. All terminase subunits are essential for DNA packaging, but the roles of individual subunits are unclear. Until now, no viral protein has been identified that binds to the sites known to be required for initiation of the DNA-cleavage/packaging reaction. We have recently made a significant advance in purifying and characterizing pUL33. In preliminary data, we show that pUL33 binds to the pac2 sequence and speculate that this serves to initiate the DNA-cleavage/packaging process. Using methods developed in our laboratory, we propose to (i) characterize the DNA-binding activities of pUL33, (ii) map the functional domains of pUL33, and (iii) test whether the DNA-binding activity plays an important role in cleavage of DNA in infected cells. These studies will directly address an important issue in the field: how the terminase recognizes packaging signals in the viral genome to initiate the DNA-cleavage / packaging processes. In addition, studies on the mechanism of DNA-cleavage/ packaging may lead to development of novel anti-herpes virus strategies.