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

单纯疱疹病毒65K DNA结合蛋白(65K DBP) 1型病毒(单纯疱疹病毒L)已被证明是由UL42编码的，并且是 来源依赖的DNA所需的仅有的七个病毒基因之一 复制。一种温度敏感(Ts)突变体，含有一个 65K DBP基因的损伤不能在 不允许的温度，证实了65K DBP在 体内病毒DNA复制。但是，的确切功能是 65K DBP及其与其他复制蛋白的相互作用 仍然是未知的，也是这个项目的长期目标。由于 它有能力编码病毒所需的许多蛋白质 DNA复制，单纯疱疹病毒提供了一种遗传上易于处理的模型系统 研究真核生物体内的DNA复制。此外， 包括65K DBP在内的7个复制基因中的每一个都是 有效的抗病毒目标。在短期内，我们将研究 65K DBP的DNA结合的通用模板特异性，以及 确定其刺激POL的能力是否源于模板 效果，速度或过程的增加，能力的增加 POL循环，或促进滞后的链合成。我们会 通过以下方式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，对病毒DNA聚合酶(Poll)的刺激， 核定位和依赖于来源的DNA复制。 最初，这些结构域中的每一个都将使用 65K DBP基因中的哪些嵌套突变已经被设计出来。 对于DNA结合和POL的刺激，质粒将被反式- 在体外切割和翻译以产生突变蛋白。瞬变 细胞的转染分析将被用来确定 突变的质粒表达65K的DBP，能够定位在 核，并能够支持来源依赖的DNA合成。 一旦确定，域将按站点(OGO)进一步本地化- 定向诱变。一种细胞系将被构建成能够 支持65K DBP基因缺陷病毒的生长和 基因工程突变将被转移到一个完整的病毒中 基因组。分离的病毒突变株将被测试其效果 病毒复制和复制能力上的突变 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.