DNA- PROTEIN INTERACTIONS IN HERPES VIRUSES

疱疹病毒是一种大型DNA病毒，其中几种会感染人类细胞，导致无数疾病，有些是严重的。抗击这些病毒需要更多地了解其复制周期的机制。这项研究计划的一个主要重点将继续是结合使用生物化学和定量电子显微镜来可视化和分析HSV-1复制所涉及的结构。三个相互关联的焦点区域是：1)在Oris形成的启动复制的复合体，2)由纯化的HSV-1蛋白在体外重组的移动复制叉的结构，以及3)分析疱疹病毒蛋白驱动的重组路径，因为它们与复制的启动有关。所有的蛋白质，包括单链结合蛋白ICP8、起始结合蛋白UL9、解旋酶-Primase(UL5/8/52)和聚合酶复合体(UL30/42)都是以纯化形式存在的。 将采用新的有效的EM方法，包括冷冻EM和脂质结晶，一种名为甘油喷雾/低电压EM的新制备方法，以及用于定位多蛋白复合体中单个蛋白质的新型纳米生物探针。此外，两种不同的亲和陷阱方法将被用来定量地从感染细胞中分离复制的疱疹DNA。这将允许对感染周期中细胞中存在的DNA形式进行第一次定量分析。在目标I中，将继续研究Oris中涉及ICP8和UL9蛋白的复合体的结构。在体外完全重组的移动式HSV-1复制叉的结构将首次被EM检查。在AIM II中，将研究ICP8和UL5/8/52以及ICP8和UL12催化的重组途径，并研究它们启动复制的能力。在目标III中，将对ICP8蛋白形成的细丝进行高分辨率EM分析，因为这些细丝与由 重组酶，如RecA。在AIM IV中，将使用Lac抑制物亲和力陷阱和RecA蛋白插入的新型生物素-补骨脂素陷阱从感染细胞中分离复制的疱疹病毒DNA进行EM分析。这些研究将在巴琴海默实验室进行。最后，Aim V将与Kenney实验室合作检查EBV起源复合体，并研究EBV EBNA-1与人端粒结合蛋白之间的相互作用。

Herpes viruses are large DNA viruses several of which infect human cells to cause a myriad of diseases some, severe. Combating these viruses will require knowing more about the mechanism of their replication cycles. A major emphasis of this research program will continue to be the combined use of biochemistry and quantitative electron microscopy to visualize and analyze the structures involved in HSV-1 replication. Three interlocking areas of focus are: 1) the complexes formed at oriS which initiate replication, 2) the structure of the moving replication fork reconstituted in vitro from the purified HSV-1 proteins, and 3) analysis of recombinational pathways driven by the Herpes proteins as they relate to the initiation of replication. All proteins including the single strand binding protein ICP8, the origin binding protein UL9, the helicase-primase (UL5/8/52, and the polymerase complex (UL30/42) are in hand in purified form. New powerful EM methods will be employed including cryoEM and lipid crystallization, a new preparative method termed glycerol spray/low voltage EM, and novel nano-scale biopointers used to localize single proteins in multiprotein complexes. In addition, two different affinity trap.approaches will be used to quantitatively isolate replicating Herpes DNA from infected cells. This will allow the first quantitative analysis of the DNA forms present in the cell during the infection cycle. In Aim I, the structure of complexes at oriS involving ICP8 and UL9 proteins will continue to be investigated. The architecture of a moving HSV-1 replication fork fully reconstituted in vitro will be examined for the first time by EM. In Aim II, recombinational pathways catalyzed by the ICP8 and UL5/8/52 and also ICP8 and UL12 will be examined and their ability to initiate replication studied. In Aim III, high resolution EM analysis of filaments formed by the ICP8 protein will be carried out as these filaments share properties with ones formed by recombinases such as RecA. In Aim IV a lac represser affinity trap and a novel biotin-psoralen trap inserted by RecA protein will be used to isolate replicating Herpes DNA from infected cells for EM analysis. These studies will be done with the Bachenheimer laboratory. Finally Aim V in collaboration with the Kenney laboratory EBV origin complexes will be examined and the interaction between EBV EBNA-1 and the human telomere binding proteins studied.