Intra-molecular interactions control papillomavirus E1 function.

描述(申请人提供)：乳头瘤病毒现在被认为是人类疾病中非常重要的致病因素。作为它们正常生命周期的一部分，这些病毒感染和转化上皮细胞，导致良性肿瘤，频率很低但很高，可能会变成恶性。更深入地了解一般的生命周期，特别是DNA复制，对于了解疾病及其传播，并最终制定有效的治疗措施至关重要。尽管最近开发的疫苗有望减少这种疾病的流行，但在可预见的未来，由于大量已经感染的人，乳头瘤病毒感染仍将是一个令人敬畏的问题。因此，诸如病毒生命周期的小分子抑制剂之类的治疗选择是可取的。与其他致病病毒相比，在确定乳头瘤病毒小分子干预靶点方面投入的努力非常有限。多年来，我们的目标一直是对乳头瘤病毒DNA复制装置建立足够详细的了解，以确定可以作为小分子干预目标的分子相互作用。我们和其他人的工作现在已经到了可以精确定位病毒复制机制的特定方面的地步，这些方面代表了小分子干扰病毒复制的看似合理的目标。在特定的目标1中，我们建议表征构成这种潜在靶点的乳头瘤病毒E1启动子蛋白中的4个分子内相互作用。我们将对这些相互作用进行生化和遗传学分析，以确定它们的特定功能、它们在乳头瘤病毒家族中的保守程度，以及它们是否在病毒生命周期中是必不可少的。这些分子内相互作用的一个有趣的方面是，它们由蛋白激酶CK2的磷酸化控制。在特定的目标2中，我们将确定病毒E1和E2蛋白的磷酸化在控制病毒DNA复制中所起的作用。我们的结果表明，这两种蛋白的磷酸化会导致生化活性的显着变化，包括DNA结合活性的丧失。我们将研究磷酸化事件导致E1和E2生化变化的机制，以及这些磷酸化事件在病毒生命周期中发挥的作用。这些研究将导致 极大地提高了对乳头瘤病毒如何调控DNA复制启动的理解。他们还将提供关于E1中的分子内相互作用是否可以发展为乳头瘤病毒感染的小分子干预目标的信息。

DESCRIPTION (provided by applicant): Papillomaviruses are now understood to be very important causal agents in human disease. As part of their normal life cycle, these viruses infect and transform cells in the epithelium causing benign tumors that with a low but significant frequency can become malignant. A deeper understanding of the life cycle in general, and DNA replication in particular, is of critical importance for the understanding of the disease, its transmission and ultimately for the development of effective therapeutic measures. Although recently developed vaccines promise to reduce the prevalence of this disease, for the foreseeable future papillomavirus infections will still present a formidable problem due to the large number of already infected individuals. Thus treatment options such as small molecule inhibitors of the viral life cycle would be desirable. Compared to other disease causing viruses very limited effort has been invested in identifying targets for small molecule intervention in papillomaviruses. Our goal has for many years been to establish a sufficiently detailed understanding of the papillomavirus DNA replication apparatus to identify molecular interactions that could serve as targets for small molecule intervention. Work by us and others has now reached the point where we can pinpoint particular aspects of the viral replication machinery that represent plausible targets for small molecule interference with viral replication. In Specifi Aim 1, we propose to characterize 4 intra-molecular interactions in the papillomavirus E1 initiator protein that constitute such potential targets. We will analyze these interactions biochemically and genetically to determine their specific functions, their degree of conservation within the papillomavirus family, and whether they are essential in the viral life cycle. An interesting aspect of these intra- molecular interactions is that they are controlled by phosphorylation by the protein kinase CK2. In Specific Aim 2, we will determine the role that phosphorylation of the viral E1 and E2 proteins play in the control of viral DNA replication. Our results show that phosphorylation of these two proteins result in dramatically altered biochemical activities including loss of DNA binding activity. We will investigate the mechanism by which the phosphorylation events cause biochemical changes in the E1 and E2 and what function these phosphorylation events serve in the viral life cycle. These studies will result in a greatly improved understanding of how papillomaviruses regulate initiation of DNA replication. They will also provide information about whether intra- molecular interactions in E1 can be developed into targets for small molecule intervention in papillomavirus infections.