HSV PROTEIN NUCLEOTIDYLATION AND LYTIC INFECTION

HSV 蛋白核苷酸化和裂解感染

• 批准号: 2517306
• 负责人: John A. Blaho
• 金额: $ 10.56万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2517306
• 关键词: casein kinase; genetic mapping; herpes simplex virus 1; posttranslational modifications; recombinant virus; site directed mutagenesis; tissue /cell culture; transfection; virus infection mechanism; virus protein

Lytic herpes simplex virus (HSV) infections in neonates and immunocompromised patients result in high morbidity an mortality. Protein nucleotidylation is a novel posttranslational modification which may act as a means to regulate lytic HSV infection. Eight HSV proteins were previously shown to be nucleotidylated in vitro in isolated nuclei from infected cells, including the four alpha regulatory proteins and four late gene products. The specific aims of this project are: (i) to determine whether HSV protein nucleotidylation occurs inside cells during viral infection and (ii) to determine what function this modification plays during th HSV replicative cycle. The hypothesis to be tested is that nucleotidylation occurs at distinct sites on the target viral protein and that it is essential for the function of theses proteins. To determine whether nucleotidylation occurs inside cells, cells will be metabolically labeled with tritiated nucleotide prior to infection with HSV. The viral proteins labeled in these cells will then be purified and their modification will be characterized biochemically. The viral protein modification sites will be identified by mapping the targets in fusion proteins that contain portions of the viral polypeptides. Site-directed mutations in the fusion proteins will be made to determine the specific amino acids required for modification. Initially, the focus will be on three representative viral proteins. Once the target modification sites are identified, point mutations will be made at these sites in the genes. The mutated genes will be placed back into the virus by recombination and the growth of the resulting viruses will be studied. Specifically, the levels of viral DNA, RNA and protein for each of the recombinants will be measured. Human casein kinase II (CKII) was implicated in the nucleotidylation of the viral alpha regulator ICP22. To determine the role that CKII plays in this process, it will be purified to homogeneity and its novel activity will be characterized biochemically. Nucleotidylation of the other alpha regulatory factors requires a viral component which is made late in infection. This component will be purified to the extent that a partial amino acid sequence can be obtained and the gene encoding the factor may be identified. These studies will help in determining whether nucleotidylation is a necessary process in the replication of HSV. In the long term, this research will help define the molecular basis for regulation of the life-cycle of this important human pathogen.

裂解疱疹病毒（HSV）在新生儿和 免疫功能低下的患者导致高发病率是死亡率。 蛋白质 核苷酸化是一种新型的翻译后修饰，可以充当 调节裂解HSV感染的一种手段。 八个HSV蛋白是 先前证明是在分离的核中体外被核苷酸化的 感染的细胞，包括四种α调节蛋白和四个晚期 基因产品。 该项目的具体目的是：（i）确定 HSV蛋白是否在病毒期间发生在细胞内部 感染和（ii）确定这种修饰的功能 在HSV复制周期中。 要检验的假设是 核苷酸化发生在靶病毒蛋白上的不同位点， 这对于这些蛋白质的功能至关重要。 确定 核苷酸是否发生在细胞内，细胞将是代谢 在感染HSV之前，用tri的核苷酸标记。 病毒 然后将标记在这些细胞中标记的蛋白质纯化，并将其纯化 修饰将以生化为特征。 病毒蛋白 修改站点将通过在融合中映射目标来识别 包含一部分病毒多肽的蛋白质。 站点定向 将制作融合蛋白中的突变以确定特定 修饰所需的氨基酸。 最初，重点将开始 三种代表性病毒蛋白。 一旦目标修改站点 鉴定出，将在基因的这些位点进行点突变。 突变的基因将通过重组和 将研究所得病毒的生长。 具体来说， 每种重组剂的病毒DNA，RNA和蛋白质水平都将是 测量。 人酪蛋白激酶II（CKII）与 病毒α调节剂ICP22的核苷酸化。 确定角色 CKII在此过程中扮演的角色将被纯化为同质性及其 新活性将以生化为特征。 核苷酸的化 其他α调节因素需要一个病毒成分 感染后期。 该组件将被纯化以至于 可以获得部分氨基酸序列，并编码编码的基因 可以识别因子。 这些研究将有助于确定是否 核苷酸化是复制HSV的必要过程。 在 长期，这项研究将有助于定义分子基础 调节这种重要人类病原体的生命周期。