HSV-1 PROCESSING AND PACKAGING GENES

HSV-1 加工和包装基因

• 批准号: 2390419
• 负责人: SANDRA K WELLER
• 金额: $ 16.28万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390419
• 关键词: Baculoviridae; antiserum; capsid; confocal scanning microscopy; genetic recombination; herpes simplex virus 1; immunofluorescence technique; immunoprecipitation; monoclonal antibody; mutant; nuclease; protein biosynthesis; protein structure function; recombinant DNA; tissue /cell culture; transfection /expression vector; virion; virus DNA; virus assembly; virus genetics; virus protein

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.

单纯疱疹病毒在人群中流行并造成 对于多种临床疾病，其中一些是危及生命的， 尤其是免疫功能低下的个体或新生儿。疱疹 1 型单纯病毒 (HSV-1) 基因组包含三个复制起点 并编码病毒基因组必需的七个病毒基因 体内复制。然而病毒DNA复制的实际模式和 重组在此过程中的作用尚不清楚。甚至更少 病毒基因组成熟的过程已广为人知 衣壳化。该提案的目的是提供更好的 通过以下方式了解基因组加工和包装的事件 对七种病毒基因进行遗传和生化分析 参与这些过程。初步证据表明 UL12， 碱性核酸酶在基因组成熟和 衣壳化。 UL12 中的无效突变体合成病毒 DNA 并启动 裂解和包装事件；然而，含有衣壳的DNA 积累在细胞核中，不成熟进入细胞质。其他六个 正在研究的病毒基因（UL6、UL15、UL25、UL28、UL32 和 UL33）已被 涉及不同阶段的基因组切割和包装。突变体 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，但无法切割或包装病毒基因组 在非许可条件下生长。 第一个目标是产生必要的试剂来进行 后续目标；这些包括每个表达的构建体 多种表达系统中的蛋白质以及 特定抗血清的表征。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。现有的大部分 HSV 和伪狂犬病 (PRV) 这些基因的突变体是温度 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， 对于分离每个基因中有效的无效突变体非常重要。第三个目标 是确定假定的加工/包装基因的位置 在病毒粒子内和受感染的细胞内。具体来说，我们计划使用 间接免疫荧光和共聚焦显微镜来表征 细胞内定位既相互相关又与 关于复制和衣壳蛋白复合物。在第四个目标中 七种蛋白质之间假定的蛋白质-蛋白质相互作用将是 通过生化和遗传学方法进行分析。长期目标包括 分析每个假定的加工/包装蛋白以预测 ATP 酶、终止酶和 DNA 结合等活动 每个基因的详细结构功能分析。预计 对加工/包装蛋白质的分析不仅可以提供 了解其作用机制，但也可能导致 开发新的抗病毒治疗策略。