HSV-1 PROCESSING AND PACKAGING GENES

单纯疱疹病毒在人群中流行，并与 对于各种临床疾病，其中一些是危及生命的， 尤其是在免疫功能受损的个体或新生儿中。疱疹 单纯疱疹病毒1型(HSV-1)基因组包含三种复制来源 并编码7个病毒基因，这些基因对病毒基因组至关重要 体内复制。然而，病毒DNA复制的实际模式和 重组在这一过程中的作用还不是很清楚。更少 众所周知，病毒基因组成熟和成熟的过程 封装。这项提议的目标是提供一个更好的 通过对基因组加工和包装事件的理解 已分离的7个病毒基因的遗传和生化分析 与这些过程有牵连。初步证据表明，UL12， 碱性核酸酶在基因组成熟和分化过程中起重要作用 封装。UL12中的零突变体合成病毒DNA并引发 分裂和包装事件；然而，包含衣壳的DNA 在细胞核内积聚，不成熟进入细胞质。其他六个人 正在研究的病毒基因(UL6、UL15、UL25、UL28、UL32和UL33)已经被 牵涉到不同阶段的基因组切割和打包。突变者 这些基因中的每一个都有一个共同的表型：它们能够进行 病毒DNA合成，但在以下情况下无法切割或打包病毒基因组 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 后续目标；这些目标包括每个目标的表达的构造 蛋白质在各种表达系统中的表达和生成 特异性抗血清的特性。第二个目标是隔离或 进一步鉴定每个基因中的零突变。大多数现有的 HSV和伪狂犬病(PRV)的这些基因中的突变是温度 敏感(Ts)；然而，由于ts突变体的潜在困难 如泄漏和干扰野生型等位基因的功能，它 对于在每个基因中分离有效的零突变是很重要的。第三个目标 是确定可能的加工/包装基因的位置 在病毒粒子和受感染的细胞内。具体来说，我们计划使用 间接免疫荧光和共聚焦显微镜表征 细胞内相互定位和相互定位 关于复制和衣壳蛋白复合体。第四个目标 这七种蛋白质之间可能存在的蛋白质-蛋白质相互作用 通过生化和遗传学方法进行分析。长期目标包括 检测每一种假定的加工/包装蛋白质以预测 ATPase、终止酶和DNA结合等活动，并执行 对每个基因进行了详细的结构和功能分析。预计 对加工/包装蛋白质的分析不仅将提供 了解他们的行动机制，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.