Role ofK8 bZip Protein in KSHV Lytic DNA Replication

K8 bZip 蛋白在 KSHV 裂解性 DNA 复制中的作用

• 批准号: 7021450
• 负责人: YAN YUAN
• 金额: $ 30.96万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021450
• 关键词: DNA replication; DNA replication origin; Kaposi' s sarcoma; autoradiography; binding sites; gene expression; genetic library; genetic mapping; genetic regulatory element; human herpesvirus 8; immunoprecipitation; mass spectrometry; molecular cloning; nucleic acid sequence; plasmids; protein protein interaction; protein purification; protein structure; protein structure function; site directed mutagenesis; transfection; transport proteins; virus DNA; virus protein; virus replication

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus8 (HHV-8), is a human tumor virus which is associated with Kaposi' s sarcoma and several lymphoproliferative diseases. Phylogenetic analysis of KSHV genomic sequence revealed that KSHV belongs to the gamma-herpesvirus family, which characteristically establishes latent infection in lymphoid cells. When latency is disrupted, viruses can switch to lytic life cycle. In general, herpesviruses utilize different origins of replication during latent versus lytic infection. Latent DNA replication depends on the cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin-binding protein (OBP) that recruits the core replication machinery. Although six core replication proteins have been identified in KSHV, an OBP and the ori-Lyt of KSHV were unidentified. Recently, some progresses were made in our laboratory. (i) two regions in the KSHV genome, between K4.2 and K5 and between K12 and ORF71, were found to be able to serve as origins for lytic cycle-specific DNA replication; (ii) a KSHV-encoded bZip protein K8 was found to bind to a sequence within KSHV ori-Lyt by an in vitro binding site selection assay. These two discoveries opened an avenue to study the mechanism of KSHV lytic DNA replication. In the proposed studies, we will investigate the mechanism of KSHV lytic DNA replication. Emphasis will be placed on the role of K8 bZip protein in the lytic-origin dependent DNA replication. (1) KSHV ori-Zyt domain will be dissected using site-directed mutagenesis to define cis-acting requirements for viral lytic replication and map K8 binding element within the ori-Lyt. (2 ) Since the function of K8 in viral lytic DNA replication appears to be mediated through interacting with the core viral replication proteins, revelation of interaction of K8 with other proteins becomes of paramount importance in understanding the role of K8 in KSHV DNA replication. We will attempt to identify the viral or cellular proteins that are associated with K8. (3) The functional role(s) of K8 in KSHV lytic DNA replication and other events in lytic cycle will be determined by using transient DNA replication assay and by blocking K8 gene expression using the external guide sequence (EGS) technique. The proposed studies will provide an insight into KSHV lytic cycle replication.

描述（由申请人提供）：Kaposi的肉瘤相关疱疹病毒（KSHV），也称为人疱疹病毒8（HHV-8），是一种与Kaposi S肉瘤和几种淋巴细胞增生性疾病有关的人类肿瘤病毒。 KSHV基因组序列的系统发育分析表明，KSHV属于γ-HERPESVIRUS家族，该家族的特征在于淋巴样细胞中的潜在感染。当潜伏期中断时，病毒可以切换到裂解生命周期。通常，疱疹病毒在潜在感染与裂解期间利用复制的不同起源。潜在的DNA复制取决于细胞DNA复制机制，而裂解循环DNA复制需要病毒编码的复制蛋白。在裂解DNA复制中，裂解起源（ori-Lyt）受募集核心复制机制的病毒指定起源结合蛋白（OBP）结合。尽管在KSHV中已经确定了六种核心复制蛋白，但KSHV的OBP和ORI-Lyt尚不确定。最近，我们的实验室取得了一些进展。 （i）发现KSHV基因组中的两个区域，K4.2和K5之间以及K12和ORF71之间的两个区域，可以作为裂解周期特异性DNA复制的起源； （ii）通过体外结合位点选择测定法，发现KSHV编码的BZIP蛋白K8与KSHV Ori-Lyt内的序列结合。这两个发现开辟了一条研究KSHV裂解DNA复制机制的途径。在拟议的研究中，我们将研究KSHV裂解DNA复制的机理。将重点放在K8 BZIP蛋白在依赖性的DNA复制中的作用。 （1）将使用定位的诱变来解剖KSHV Ori-Zyt结构域，以定义对病毒裂解复制的顺式作用要求，并在Ori-Lyt中绘制K8结合元件。 （2）由于K8在病毒裂解DNA复制中的功能似乎是通过与核心病毒复制蛋白相互作用而介导的，因此K8与其他蛋白质的相互作用的启示在理解K8在KSHV DNA复制中的作用至关重要。我们将尝试确定与K8相关的病毒或细胞蛋白。 （3）K8在KSHV裂解DNA复制中的功能作用将通过使用瞬态DNA复制测定法和使用外部指南序列（EGS）技术阻止K8基因表达来确定裂解周期中的其他事件。提出的研究将提供对KSHV裂解周期复制的见解。