DNA- PROTEIN INTERACTIONS IN HERPES VIRUSES

疱疹病毒是一种大型DNA病毒，其中一些感染人类细胞，导致无数疾病，有些很严重。抗击这些病毒需要更多地了解它们的复制周期机制。该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复制的结构。三个相互关联的重点领域是：1)在口腔处形成的启动复制的复合物，2)从纯化的HSV-1蛋白在体外重建的移动复制叉的结构，以及3)分析由疱疹蛋白驱动的重组途径，因为它们与复制的启动有关。所有的蛋白，包括单链结合蛋白ICP8、起始结合蛋白UL9、解旋酶引物酶（UL5/8/52）和聚合酶复合物（UL30/42）都以纯化的形式存在。

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.