FUNCTIONAL DOMAINS OF THE HSV-1 65K DNA BINDING PROTEIN

单纯疱疹病毒的65 KDBP 1型病毒（HSV-1）已显示由UL 42编码， 只有七个病毒基因需要来源依赖的DNA之一， 复制的 一种温度敏感（ts）突变体， 65 K DBP基因中的损伤不能合成病毒DNA， 非允许温度，证实了65 K DBP在 体内病毒DNA复制。 然而， 65 K DBP及其如何与其他复制蛋白相互作用 这是一个未知数，也是这个项目的长期目标。 由于 它能够编码许多病毒所需的蛋白质， DNA复制，HSV提供了一个遗传上易于处理的模型系统 用于研究真核生物的DNA复制 此外，委员会认为， 包括65 K DBP在内的七个复制基因中的每一个都是 有效的抗病毒靶点。 在短期内，我们会研究 65 K DBP的DNA结合的一般模板特异性，和 确定其刺激pol的能力是否是由于模板 影响，速率或持续合成能力的增加， 或促进滞后链的合成。 我们将 确定蛋白质的各种功能结构域， 确定蛋白质的哪些区域是特别需要的 对于DNA结合，刺激病毒DNA聚合酶（pol）， 核定位和来源依赖的DNA复制。 最初，这些结构域中的每一个都将使用质粒进行研究， 65 K DBP基因中的哪些巢式突变已被工程化。 为了DNA结合和刺激pol，质粒将被转移到 在体外转录和翻译以产生突变蛋白。 瞬态 细胞的转染分析将用于确定是否 突变的质粒表达能够定位于细胞内的65 K DBP。 细胞核和能够支持起源依赖性DNA合成。 一旦确定，结构域将进一步定位的网站（寡核苷酸）- 定向诱变 将构建能够 支持具有65 K DBP基因缺陷的病毒的生长， 工程突变将被转移到一个完整的病毒 基因组 将检测分离的病毒突变体的作用， 病毒复制能力的突变， 在体内合成病毒DNA。

The 65 kilodalton DNA binding protein (65K DBP) of herpes simplex virus type 1 (HSV-l) has been shown to be encoded by UL42 and is one of only seven viral genes required for origin-dependent DNA replication. A temperature sensitive (ts) mutant containing a lesion in the 65K DBP gene does not synthesize viral DNA at the nonpermissive temperature, confirming the role of the 65K DBP in viral DNA replication in vivo. However, the exact function of the 65K DBP and how it interacts with the other replication proteins remains unknown and is the long term goal of this project. Due to its ability to encode so many of the proteins required for viral DNA replication, HSV provides a genetically tractable model system for studying DNA replication in a eukaryotic organism. Moreover, each of the seven replication genes, including the 65K DBP, are valid antiviral targets. In the short term, we will examine the general template specificity for DNA binding of the 65K DBP, and determine whether its ability to stimulate pol is due to template effects, increase in rate or processivity, increase in the ability of pol to cycle, or promotion of lagging strand synthesis. We will determine the various functional domains of the protein by determining which regions of the protein are specifically required for DNA binding, stimulation of the viral DNA polymerase (pol), nuclear localization, and origin-dependent DNA replication. Initially each of these domains will be studied using plasmids in which nested mutations in the 65K DBP gene have been engineered. For DNA binding and stimulation of pol, plasmids will be tran- scribed and translated in vitro to yield mutant protein. Transient transfection analysis of cells will be used to determine whether mutated plasmids express a 65K DBP capable of localizing in the nucleus and capable of supporting origin-dependent DNA synthesis. Once identified, domains will be further localized by site(oligo)- directed mutagenesis. A cell line will be constructed capable of supporting the growth of virus with defects in the 65K DBP gene and engineered mutations will be transferred into an intact viral genome. Isolated viral mutants will be tested for the effect of the mutation on the ability of the virus to replicate and synthesize viral DNA in vivo.