Molecular basis and regulation of the DNA binding activity of KSHV LANA

描述（由申请人提供）：卡波西氏肉瘤相关疱疹病毒（KSHV）是几种人类疾病的病原，包括卡波西氏肉瘤、原发性积液性淋巴瘤和多中心Castleman病的一个亚群。KSHV感染存在两种状态：非生产性潜伏状态和增殖裂解状态。在潜伏期，病毒基因表达集中在那些编码防止宿主免疫反应和维持潜伏期状态所需产物的基因上。为了确保病毒感染在细胞分裂后持续存在，病毒基因组作为一个片段循环，并通过蛋白质潜伏期相关核抗原（LANA）拴在宿主染色体上。LANA是一种多功能蛋白，在维持小体的通道以及抑制裂解再激活和宿主免疫反应中起关键作用。LANA的n端与宿主核小体相互作用，而c端将序列特异性地与病毒片段的一部分结合。与核小体结合的n端肽的结构先前已经解决；然而，在c端没有结构信息。为了了解LANA如何介导这种序列特异性相互作用，我们建议确定LANA DNA结合域的三维晶体结构，包括单独的和与其DNA结合位点结合的。利用这些结构信息，我们将在我们认为对序列特异性和DNA结合功能重要的区域创建突变。此外，我们已经在c端发现了新的丝氨酸磷酸化位点，这些位点被认为在与细胞蛋白的相互作用中发挥作用，并可能对DNA结合产生影响。我们建议使这些丝氨酸的突变来废除或模仿磷酸化的构成状态。我们将利用先前开发的检测方法来检测这些突变的DNA结合、插曲体维持和基因组复制。从这些研究中获得的信息将为开发LANA的小分子抑制剂以及理解翻译后修饰在DNA结合和病毒生命周期中的作用提供基础。

DESCRIPTION (provided by applicant): Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of several human diseases, including Kaposi's sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman's disease. KSHV infections exist in two states: a non-productive latent state and a proliferative lytic state. During latency, viral gene expression focuses on those genes that encode products required for preventing a host immune response and maintaining a state of latency. To ensure that viral infection persists after cell division, the viral genome is circularized as an episome and tethered to the host chromosome by the protein latency-associated nuclear antigen (LANA). LANA is a multi-functional protein that plays a pivotal role in maintaining passage of the episome as well as repressing lytic reactivation and host immune response. The N-terminus of LANA interacts with a host nucleosome while the C-terminus binds sequence specifically to a portion of the viral episome. The structure of an N-terminal peptide bound to the nucleosome has previously been solved; however, no structural information is available on the C-terminus. To understand how LANA mediates this sequence specific interaction we propose to determine the three-dimensional crystal structure of the LANA DNA binding domain both alone and bound to its DNA binding site. Utilizing this structural information we will then create mutations in regions that we identify to e important for sequence specificity and DNA binding function. Also, we have identified novel serine phosphorylation sites within the C-terminus that have been suggested to play a role in interactions with cellular proteins and may have an effect on DNA binding. We propose to make mutations of these serines to either abrogate or mimic a constitutive state of phosphorylation. We will utilize previously developed assays to test these mutations for DNA binding, episome maintenance, and genome replication. The information obtained from these studies will provide a foundation for the development of small-molecule inhibitors of LANA as well as an understanding of the role of post-translational modifications in DNA binding and the viral life cycle. PUBLIC HEALTH RELEVANCE: Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is a human viral pathogen that is the causative agent of several lymphomas. The KSHV genome is persistent in infected cells and is directly attached to the host chromosome. The goal of this project is to determine the three-dimensional structure of the viral genome binding portion of the protein LANA, which is responsible for attaching the viral genome to the host chromosome and to understand how modifications to this protein affect its DNA binding function.