HSV REPLICATION, EARLY ENZYMES AND LATENCY

本项目研究病毒DNA复制蛋白在 单纯疱疹病毒与哺乳动物神经系统的相互作用 特别是病毒潜伏期。 潜伏期是一种迷人的生物学现象， 病毒的特性，因为潜伏病毒是反复发作的来源， 疾病，是一个重要的临床特征。 HSV潜伏期信息 与靶向复制蛋白的抗病毒药物高度相关， 和疫苗，并可能最终允许设计的代理人治愈 疱疹病毒感染 该研究的目的是：1.量化HSV DNA的构型 在潜伏期期间，通过脉冲场凝胶电泳结合聚合酶 链反应（PCR）和连接介导的PCR。 这些高度敏感 方法将被应用到几个问题，包括配置 神经节中的野生型和突变型HSV DNA。 第二个目标是利用这些 方法，以遵循HSV DNA的构型，在建立 潜伏期，特别是胸苷激酶（TK）突变体。 定量RNA PCR cDNA克隆将用于HSV基因的定量和表征 在潜伏期建立期间表达。 的舞台 在反应期间HSV复制被阻断的感染周期， TK或核糖核苷酸还原酶（RR）缺失或抑制时的潜伏期 将确定（目标3）。 感染tk或rr无效突变体的神经节或 在TK抑制剂存在下，将检查野生型病毒的 使用原位杂交和PCR进行病毒基因表达。 功能 HSV TK和HSV DNA聚合酶（Pol）的非必需功能， 对外周和中枢神经系统的发病机制很重要 将得到确认（目标4）。 重组病毒，其中HSV tk基因 通过插入人tk或脱氧胞苷激酶基因而失活， 将测试某些TK突变病毒的神经节复制， 再活化和神经毒性。 将检测HSV pol突变体， 确定某些POL功能是否对于 复制和发病机制。 拟议的实验应 揭示了病毒和神经系统生物学的问题， 基因表达、抗病毒药物抗性和HSV作为载体的用途 或者作为治疗

This project investigates the roles of viral DNA replication proteins in the interaction of herpes simplex virus (HSV) with the mammalian nervous system, especially virus latency. Latency is a fascinating biological property of the virus and, because latent virus is the source of recurrent disease, is an important clinical feature. Information about HSV latency is highly relevant to antiviral drugs, which target replication proteins, and vaccines, and might eventually permit the design of agents to cure herpesvirus infections. The proposed research will (aim 1) quantify configurations of HSV DNA during latency by pulsed-field gel electrophoresis coupled with polymerase chain reaction (PCR) and by ligation-mediated PCR. These highly sensitive methods will be applied to several problems including the configuration of wild type and mutant HSV DNA in ganglia. The second aim is to use these methods to follow the configurations of HSV DNA during the establishment of latency, especially by thymidine kinase (tk) mutants. Quantitative RNA PCR and cDNA cloning will be used to quantify and characterize HSV gene expression during the establishment of latency. The stage in the infectious cycle at which HSV replication is blocked during reaction from latency when TK or ribonucleotide reductase (RR) is absent or inhibited will be determined (aim 3). Ganglia infected with tk or rr null mutants or by wild type virus in the presence of TK-inhibitors will be examined for viral gene expression using in situ hybridization and PCR. Functions of HSV TK and non-essential functions of HSV DNA polymerase (Pol) that are important for pathogenesis in the peripheral and central nervous systems will be ascertained (aim 4). Recombinant viruses, in which the HSV tk gene is inactivated by insertion of a human tk or deoxycytidine kinase gene, and certain tk mutant viruses will be tested for ganglionic replication and reactivation and for neurovirulence. HSV pol mutants will be tested to determine if certain pol functions are particularly important for replication and pathogenesis in the brain. The proposed experiments should shed light on issues of virus and nervous system biology, regulation of gene expression, antiviral drug resistance, and the use of HSV as a vector or as therapy.