Role ofK8 bZip Protein in KSHV Lytic DNA Replication

描述(申请人提供)：卡波西肉瘤相关疱疹病毒(KSHV)，又称人类疱疹病毒8(HHV-8)，是一种人类肿瘤病毒，与卡波西S肉瘤和多种淋巴增生性疾病有关。对KSHV基因组序列的系统发育分析表明，KSHV属于伽马疱疹病毒家族，其特征是在淋巴细胞中潜伏感染。当潜伏期被破坏时，病毒可以切换到裂解生命周期。一般来说，疱疹病毒在潜伏感染和裂解感染期间利用不同的复制来源。潜伏的DNA复制依赖于细胞DNA复制机制，而裂解周期DNA复制需要病毒编码的复制蛋白。在裂解DNA复制中，裂解起点(Ori-Lyt)由病毒特定的原点结合蛋白(OBP)结合，该蛋白招募核心复制机制。虽然已在KSHV中鉴定出6个核心复制蛋白，但尚未鉴定KSHV的OBP和Ori-Lyt。最近，我们实验室取得了一些进展。(I)KSHV基因组中K4.2和K5之间以及K12和ORF71之间的两个区域被发现能够作为裂解循环特异性DNA复制的起点；(Ii)通过体外结合位点选择试验，发现KSHV编码的bZip蛋白K8与KSHV Ori-Lyt内的序列结合。这两个发现为研究KSHV裂解DNA复制机制开辟了新的途径。在拟议的研究中，我们将研究KSHV裂解DNA复制的机制。重点将放在K8bZip蛋白在依赖于裂解起源的DNA复制中的作用。(1)KSHV Ori-Zyt结构域将通过定点突变被切割，以确定病毒裂解复制的顺式作用要求，并将K8结合元件定位在Ori-Lyt中。(2)由于K8在病毒裂解DNA复制中的作用似乎是通过与病毒核心复制蛋白的相互作用来实现的，因此揭示K8与其他蛋白的相互作用对于了解K8在KSHV DNA复制中的作用是至关重要的。我们将尝试确定与K8相关的病毒或细胞蛋白。(3)K8在KSHV裂解过程中的功能作用(S)将通过瞬时复制实验和利用外部引导序列技术阻断K8基因的表达来确定。建议的研究将提供对KSHV裂解周期复制的洞察力。

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.