EXPRESSION AND REGULATION OF VACCINIA VIRUS LATE GENES

在感染细胞的背景下，牛痘病毒（VV）表达更多的 在晚期（即，后DNA 合成）阶段。 这些后期功能 包括结构蛋白和病毒酶，它们被包装到 子代病毒体。 到目前为止，只有有限数量的VV晚期基因被发现。 鉴定 此外，对这两种情况知之甚少。 大多数编码的晚期蛋白质的结构和功能， 调节机制（转录和转录后）， 调节其活动，以促进其 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管理的 大小，我们选择了几个有代表性的VV晚期蛋白进行详细的 研究是为了让我们实现研究的双重目标， 学习过程中VV编码的晚期基因产物的表达和调节 一些关于蛋白质定位，病毒， 宿主细胞相互作用，以及翻译后蛋白的作用 修饰（ADP-核糖基化和酰化）蛋白质功能。 的 要研究的特定基因和要进行的实验 包括：L 65（Rifr）定点诱变、嵌合基因融合， 免疫分析将用于研究这种蛋白质的功能， 鉴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 装配过程中的工厂;N2（a-Am 1）指导遗传学和PCR辅助 基因组病毒DNA的测序将用于确认“核 参与”的N2蛋白，免疫试剂将被开发， 允许在感染期间跟踪N2蛋白，并识别潜在的 细胞靶点;A（DP-核糖基化）VP 1 -8和M（苯三酰化）VP 1 -2 - 辅基的化学性质将通过蛋白质分析 化学和HPLC技术，将鉴定VV底物蛋白 通过无细胞翻译和免疫测定方法，肽图将B 用于鉴定修饰位点，定向遗传学用于 评估是否需要对蛋白质进行修饰， 不降低 研究VV的基本原理有两个方面。 首先，作为真核生物的模型， 系统所获得的信息应具有普遍相关性， to the regulation调节of gene基因expression表达. 此外，能够轻松地 进行定向遗传学和基因置换（标记转移）使 这种病毒系统非常适合这类研究。 二是 这项工作的结果应有助于验证与确认的持续发展， 用作疫苗的真核克隆和表达载体，或 生产生物制药产品。

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.