T-antigen Binding to the Merkel Cell Carcinoma Virus Origin

描述(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，与一种侵袭性皮肤癌有关。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研究较好的家族成员，如小鼠多瘤病毒和猿猴病毒40(SV40)的序列有很大不同。所有这些病毒的起始处都含有五聚体DNA序列GAGGC的多个副本，但MCV的起始点比研究得更好的病毒包含更多的副本和更紧密的副本。在多瘤病毒中，识别起源、DNA融化和招募启动病毒基因组复制的宿主细胞蛋白所需的只是一种单一的蛋白质--大T抗原。在此之前，我们已经确定了与SV40的GAGGC序列结合的T抗原域的晶体结构。基于这些结构，我们已经建立了T抗原结合MCV起源的初步模型。这些模型表明，当与MCV起源的DNA结合时，T抗原域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。这项拟议的工作旨在确定预测的相互作用是否发生，并表征在MCV起源形成的T抗原-DNA复合体的结构。由于T抗原的N-末端和C-末端区域不是相关病毒体外复制所必需的，我们将集中在MCV的起源结合域和解旋酶结构域。这项拟议的工作利用了成熟的方法，包括DNA足迹、凝胶位移和大分子结晶学。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性平台。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。拟议的项目旨在了解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DNA的能力来对抗MCV和相关病毒的疗法。

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.