HSV-1 PROCESSING AND PACKAGING GENES

单纯疱疹病毒在人群中流行， 对于多种临床疾病，其中一些是危及生命的， 特别是在免疫功能低下的个体或新生儿中。疱疹 单纯病毒1型（HSV-1）基因组包含三个复制起点 并编码七个病毒基因， 体内复制然而，病毒DNA复制的实际模式和 重组在这个过程中的作用还没有得到很好的理解。更不 病毒基因组成熟的过程已被很好地理解， - -这项建议的目的是提供一个更好的 了解基因组处理和包装的事件，通过 基因和生化分析的七个病毒基因， 在这些过程中。初步证据表明，UL 12， 碱性核酸酶在基因组成熟中起重要作用， - - UL 12中的突变体合成病毒DNA并启动 切割和包装事件;然而， 在细胞核中积累，不成熟进入细胞质。其他六 研究中的病毒基因（UL 6、UL 15、UL 25、UL 28、UL 32和UL 33）已经被 在不同阶段参与基因组切割和包装。突变体 在这些基因中的每一个都有一个共同的表型：它们能够执行 病毒DNA合成，但不能切割或包装病毒基因组时， 在不允许的条件下生长。 第一个目标是产生必要的试剂来进行 随后的目标;这些包括表达每一个 蛋白质在各种表达系统中的表达，以及 特异性抗血清的表征。第二个目标是隔离或 进一步表征每个基因中的无效突变体。大多数现有 HSV和伪狂犬病（PRV）的这些基因中的突变体是温度突变体。 敏感（ts）;然而，由于ts突变体的潜在困难 例如泄漏和干扰野生型等位基因的功能， 分离每个基因中的有效无效突变体是重要的。第三个目标 是确定假定的加工/包装基因的位置 在病毒体和感染的细胞内。具体来说，我们计划使用 间接免疫荧光和共聚焦显微镜来表征 细胞内定位都相对于彼此和与 关于复制和衣壳蛋白复合物。第四个目标 这七种蛋白质之间的蛋白质相互作用将是 通过生物化学和遗传学方法分析。长期目标包括 测定每种推定的加工/包装蛋白的预测 活动，如ATP酶，终止酶，和DNA结合，并进行 每个基因的详细结构-功能分析。预计各国 对加工/包装蛋白的分析将不仅提供 了解其作用机制，但也可能导致 开发新的抗病毒治疗策略。

Herpes simplex viruses are endemic in the population and are responsible for a variety of clinical diseases some of which are life threatening, especially in immunocompromised individuals or in newborns. The herpes simplex virus type-1 (HSV-1) genome contains three origins of replication and encodes seven viral genes that are essential for viral genome replication in vivo. However actual mode of viral DNA replication and the role of recombination in this process are not well understood. Even less well understood are the processes of viral genome maturation and encapsidation. The objective of this proposal is to provide a better understanding of the events of genome processing and packaging through the genetic and biochemical analysis of seven viral genes that have been implicated in these processes. Preliminary evidence indicates that UL12, alkaline nuclease, plays an important role in genome maturation and encapsidation. Null mutants in UL12 synthesize viral DNA and initiate cleavage and packaging events; however, the DNA containing capsids which accumulate in the nucleus do not mature into the cytoplasm. The other six viral genes under study (UL6, UL15, UL25, UL28, UL32 and UL33) have been implicated in genome cleavage and packaging at a different stage. Mutants in each of these genes have a common phenotype: they are able to carry out viral DNA synthesis but are unable to cleave or package viral genomes when grown under nonpermissive conditions. The first aim is to generate the necessary reagents to carry out the subsequent aims; these include constructs for the expression of each protein in a variety of expression systems and the generation of and characterization of specific antisera. The second aim is to isolate or further characterize null mutants in each gene. Most of the existing mutants in these genes for both HSV and pseudorabies (PRV) are temperature sensitive (ts); however, because of potential difficulties with ts mutants such as leak and interference with the function of wild type alleles, it is important to isolate effective null mutants in each gene. The third aim is to establish the location of the putative processing/packaging genes within virions and within infected cells. Specifically we plan to use indirect immunofluorescence and confocal microscopy to characterize the intracellular localization both with respect to each other and with respect to replication and capsid protein complexes. In the fourth aim putative protein-protein interactions between the seven proteins will be analyzed by biochemical and genetic methods. Long term aims include assaying each putative processing/packaging protein for predicted activities such as ATPase, terminase, and DNA binding and carrying out a detailed structure-function analysis of each gene. It is anticipated that an analysis of the processing/packaging proteins will not only provide an understanding of the mechanisms of their action but may also lead to the development of novel strategies for antiviral therapy.