T-antigen Binding to the Merkel Cell Carcinoma Virus Origin

描述（由申请人提供）：默克尔细胞癌病毒（MCV）是最近发现的多瘤病毒家族成员，与侵袭性皮肤癌有关。我们对这种病毒的兴趣源于这样一个事实，即病毒复制起源的序列与得到更好研究的家族成员，如小鼠多瘤病毒和类人猿病毒40 （SV40）的序列明显不同。所有这些病毒在起源处都含有五聚体DNA序列GAGGC的多个拷贝，但MCV起源比研究得更好的病毒包含更多拷贝和更紧密间隔的拷贝。在多瘤病毒中，一个单一的蛋白质，大t抗原，是所有需要的起源识别，DNA融化和宿主细胞蛋白质的募集，启动病毒基因组的复制。之前我们已经确定了结合SV40中GAGGC序列的t抗原结构域的晶体结构。基于这些结构，我们已经生成了t抗原结合MCV起源的初步模型。这些模型表明，当t抗原结构域与MCV起源处的DNA结合时，它们之间存在显著的相互作用，但在SV40系统的结构中没有观察到这种相互作用。提出的工作旨在确定预测的相互作用是否发生，并表征在MCV起源处形成的t抗原- dna复合物的结构。由于t抗原的N端和C端区域不需要相关病毒的体外复制，因此我们将集中讨论MCV的起源结合域和解旋酶域。提出的工作利用完善的方法，包括DNA足迹，凝胶移位，和大分子晶体学。我们预计这些研究将揭示病毒复制的重要、一般特征，并为未来旨在抑制MCV复制的努力提供一个结构平台。公共卫生相关性：默克尔细胞癌病毒（MCV）是感染人类的五种与癌症相关的DNA肿瘤病毒之一。该项目旨在了解MCV蛋白（t抗原）如何与病毒DNA相互作用，从而开始复制病毒基因组的过程。我们预计，了解这一过程将导致通过阻断MCV和相关病毒复制其DNA的能力来对抗它们的治疗方法。

DESCRIPTION (provided by applicant): Merkel cell carcinoma virus (MCV) is a recently-discovered member of the polyomavirus family that is associated with an aggressive form of skin cancer. Our interest in this virus stems from the fact that the sequence of the viral origin of replication is significantly different from that of better studied family members such as murine polyomavirus and simian virus 40 (SV40). All of these viruses contain multiple copies of the pentameric DNA sequence GAGGC at the origin, but the MCV origin contains both more copies and more closely spaced copies than the better studied viruses. In polyomaviruses, a single protein, large T-antigen, is all that is required for origin recognition, DNA melting and the recruitment of host cell proteins that initiate replication of the viral genome. Previously we have determined crystal structures of the T-antigen domain that binds the GAGGC sequence from SV40. Based on these structures we have generated preliminary models of the T-antigen-bound MCV origin. These models suggest significant interaction between the T-antigen domains when bound to DNA at the MCV origin, but such interactions were not observed in structures from the SV40 system. The proposed work aims to determine if the predicted interactions take place and to characterize the structures of the T-antigen-DNA complexes formed at the MCV origin. Since the N- and C- terminal regions of T-antigen are not required for in vitro replication of related viruses, we will concentrate on the MCV origin- binding domain and the helicase domain. The proposed work utilizes well established methods including DNA footprinting, gel shifts, and macromolecular crystallography. We anticipate that these studies will both reveal important, general features of viral replication and provide a structural platform for future efforts aimed at inhibiting replication of MCV. PUBLIC HEALTH RELEVANCE: Merkel cell carcinoma virus (MCV) is one of five cancer-related DNA tumor viruses that infect humans. The proposed project aims to understand how the MCV protein, T-antigen, interacts with the viral DNA so as to begin the process of copying the viral genome. We anticipate that understanding this process will lead to therapeutics that combat MCV and related viruses by blocking their ability to replicate their DNA.