HSV REPLICATION, EARLY ENZYMES AND LATENCY

该项目研究了病毒DNA复制蛋白在 单纯疱疹病毒与哺乳动物神经的相互作用 系统，特别是病毒的潜伏期。潜伏期是一种迷人的生物学 病毒的性质和，因为潜伏病毒是复发的来源 疾病，是一种重要的临床特征。有关HSV延迟的信息 与抗病毒药物高度相关，这种药物针对复制蛋白， 和疫苗，并可能最终允许设计治疗药物 疱疹病毒感染。 拟议的研究将(目标1)量化HSV DNA的构型 脉冲场凝胶电泳法与聚合酶联用检测潜伏期 链式反应和连接介导的聚合酶链式反应。这些高度敏感的 方法将应用于几个问题，包括 神经节中的野生型和突变型HSV DNA。第二个目标是使用这些 方法根据单纯疱疹病毒DNA的构型，建立 潜伏期，尤其是胸苷激酶(Tk)突变体。定量RNA聚合酶链式反应 并将使用克隆的方法来量化和表征HSV基因 在潜伏期建立期间表达。舞台上的舞台 单纯疱疹病毒复制被阻断的感染循环 TK或核糖核苷酸还原酶(RR)缺失或抑制时的潜伏期 将被确定(目标3)。感染tk或rr零突变体或 由野生型病毒在TK抑制剂存在的情况下进行检测 用原位杂交和聚合酶链式反应检测病毒基因的表达。的功能 HSV TK和HSV DNA聚合酶(POL)的非必要功能 在外周和中枢神经系统的发病机制中起重要作用 将被确定(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，以及 将对某些tk突变病毒进行神经节复制和 重新激活和神经毒性。HSV pol突变株将进行检测以 确定某些轮询函数是否对 大脑中的复制和发病机制。建议的实验应该是 阐明病毒和神经系统生物学问题，调节 基因表达、抗病毒耐药性和单纯疱疹病毒载体的使用 或者作为一种治疗。

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.