EXPRESSION & REGULATION OF VACCINIA VIRUS LATE GENES

在受感染的细胞中，痘苗病毒(VV)表达更多 在晚期(即DNA后)有100多个病毒编码的基因产物 合成)阶段的病毒复制周期。这些后期功能 包括结构蛋白和病毒酶，它们被包装成 子代病毒粒子。到目前为止，只有有限数量的VV晚期基因 已确认身份。此外，人们对这两种情况都知之甚少 大多数编码的晚期蛋白的结构和功能 调节机制(转录和转录后) 运作以调节他们的活动，以促进他们的 参与感染性子代病毒粒子的有序组装 粒子。为了把这个复杂的问题分解成一个可处理的 大小，我们选择了几个有代表性的VV晚期蛋白进行详细的 研究是为了使我们能够实现学习的双重目标 VV编码的晚期基因产物在学习过程中的表达和调控 关于蛋白质局部化现象，病毒- 宿主细胞的相互作用以及翻译后蛋白的作用 蛋白质功能的修饰(ADP-核糖化和酰化)。这个 要研究的特定基因和要进行的实验 包括：L65(RIFR)定点突变、嵌合基因融合和 免疫分析将被用于研究这种蛋白质的功能，以 确定与体内相关的病毒和细胞因子， 以及将其定位于病毒的顺式信息的性质 组装过程中的工厂；N_2(a-Am1)指导的遗传学和聚合酶链式反应辅助 基因组病毒DNA的测序将被用来确认 N_2蛋白的参与，免疫试剂将被开发成 允许在感染期间跟踪N_2蛋白，并识别潜在的 细胞靶标；A(DP-核糖化)VP1-8和M(黄烷化)VP1-2-The 假体的化学性质将通过蛋白质进行分析 化学和高效液相技术，将鉴定VV底物蛋白 通过无细胞翻译和免疫分析方法，肽图谱将被 用来识别修饰部位，定向遗传学用来 评估蛋白质是否需要修饰才能成为 功能齐全。 研究VV的理由有两个。首先，作为真核生物的模型 系统所获得的信息应与以下方面具有普遍相关性 对基因表达的调控。此外，能够轻松地 进行定向遗传和基因置换(标记转移)使 这种病毒系统非常适合这些类型的研究。第二， 这项工作的结果应有助于VV的继续发展，因为 用作疫苗或疫苗的真核克隆和表达载体 生物制药产品的生产。

Within the context of the infected cell, vaccinia virus (VV) expresses more than 100 virus-encoded gene products during the late (i.e., post-DNA synthesis) phase of the viral replicative cycle. These late functions include both structural proteins and viral enzymes which are packaged into progeny virions. To date, only a limited number of VV late genes have been identified. Furthermore, very little is known concerning either the structure and function of most of the encoded late proteins or the regulatory mechanisms (transcriptional and posttranscriptional) which operate to modulate their activity in order to facilitate their participation in the orderly assembly of infectious progeny virion particles. In order to dissect this complex problem down to a manageable size, we have chosen several representative VV late proteins for detailed study was made to allow us to achieve the dual goals of studying the expression and regulation of VV-encoded late gene products while learning something in general about the phenomena of protein localization, virus- host cell interactions, and the role of posttranslational protein modifications (ADP-ribosylation, and acylation) in protein function. The specific genes to be studied and the experiments to be carried out include:L65(Rifr) -site-directed mutagenesis, chimeric gene fusions, and immunoassays will be used to study the function of this protein, to identify the viral and cellular factors that is associates with in vivo, and the nature of the cis-information which localizes it to the virus factory during assembly;N2 (a-Am1) - directed genetics and PCR-aided sequencing of genomic viral DNA will be used to confirm the "nuclear involvement" of the N2 protein, immunological reagents will be developed to allow tracking of the N2 protein during infection and to identify potential cellular targets;A (DP-ribosylated)VP1-8 & M(yristylated)VP1-2 - the chemical nature of the prosthetic group will be analyzed by protein chemistry and HPLC techniques, the VV substrate proteins will be identified by cell-free translation and immunoassay methods, peptide mapping will b used to identify sites of modification, and directed genetics used to assess whether modifications are required for the proteins to become functional. The rationale for studying VV are two-fold. First, as a model eukaryotic system the information obtained should be of general relevance with regard to the regulation of gene expression. Furthermore, the ability to easily carry out directed genetics and gene replacement (marker transfer) makes this viral system quite amenable to these type of studies. Second, the results of this work should facilitate the continued development of VV as an eukaryotic cloning and expression vector for use as a vaccine or the production of biopharmaceutical products.