WALLEYE DERMAL SARCOMA VIRUS GENE EXPRESSION

白眼真皮肉瘤病毒基因表达

• 批准号: 2111154
• 负责人: SANDRA L QUACKENBUSH
• 金额: $ 2.37万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-10-06 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2111154
• 关键词: WALLEYE; DERMAL; SARCOMA; VIRUS; GENE

The long term objectives of this research are to determine the mechanism responsible for induction and regression of walleye dermal sarcoma (WDS). This tumor is associated with an unusually large (13kb) retrovirus, walleye dermal sarcoma virus (WDSV). Walleye dermal sarcomas develop and regress on a seasonal basis providing a unique model to study retroviral- induced disease. Northern blot analysis and sequence information of WDSV indicates that it is a complex retrovirus which contains at least three accessory genes. The study of viral gene expression of WDSV is essential to the understanding of the regulatory mechanisms involved in tumor development and regression. The basal promoter activity of the WDSV LTR will be determined by measuring the activity of reporter gene constructs (LTR-CAT) in uninfected and WDSV-infected fish cell lines. Analysis of viral encoded functions involved in transactivation of the WDSV LTR will employ a recently developed cell culture system for WDSV infection. The consensus sequences of the WDSV LTR involved in transcriptional regulation and transactivation will be identified using LTR deletion mutants. A major aim of this proposal is to establish a transcriptional map of WDSV utilizing RT-PCR of mRNA and subsequent cloning and sequencing of these cDNAs. The identification and characterization of these accessory gene transcripts will provide the reagents necessary to conduct studies on the proteins encoded by these genes and their involvement in viral gene expression. These experiments should provide the foundation for further investigation of the role of viral accessory genes in tumorigenesis.GRANT=F32AI09301 The mechanisms by which retrovirus assembles are largely unknown. While progress has been made toward the understanding of the roles of the domains of the gag polyprotein precursor in assembly little is known about how this protein interacts with the cell during this process. Furthermore, the processes of capsid envelopment are often more poorly understood. The assembly and budding of the prototypical D-type retrovirus, Mason-Pfizer monkey virus, will be investigated. This virus offers a distinct advantage for the proposed studies since, unlike the C-type viruses, such as HIV, the processes of capsid assembly and envelopment are temporally and spatially separated in the cell. This separation will allow the examination of these two stages of assembly individually in vitro. Since preliminary experiments have shown the feasibility of in vitro translation for assembly studies this system will be employed to examine the incorporation of protein and RNA into the immature capsid. Analysis of M-PMV gag mutants and HIV/M-PMV chimeras will be performed to delineate domains necessary for efficient cytoplasmic assembly. The assembly of mutant viruses in vitro and in vivo will be compared in order to determine the influence of cellular transport mechanisms upon the process. In addition, the role of cytoplasmic components such as chaperonins in capsid assembly will be investigated. This system, will also be employed for the analysis of capsid envelopment in vitro. Again, the role of cellular components will be investigated. A detailed description of the mechanisms of retrovirus assembly will facilitate the development of therapeutic inhibitors of replication. Moreover, an in vitro assembly system will provide a convenient assay to screen such potential inhibitors.

这项研究的长期目标是确定机制 负责角膜白斑皮肤肉瘤（WDS）的诱导和回归。 该肿瘤与异常大（13KB）逆转录病毒有关， 角膜皮肤肉瘤病毒（WDSV）。 角膜皮真皮肉瘤发展和 季节性回归，提供了一个独特的模型来研究逆转录病毒 诱发疾病。 WDSV的北印迹分析和序列信息 表明它是一个复杂的逆转录病毒，至少包含三个 附件基因。 WDSV病毒基因表达的研究是必不可少的 了解肿瘤涉及的调节机制 发展和回归。 WDSV LTR的基础启动子活性 将通过测量报告基因构建体的活性来确定 （LTR-CAT）在未感染和WDSV感染的鱼类细胞系中。 分析 涉及WDSV LTR反式激活的病毒编码功能将 采用最近开发的WDSV感染细胞培养系统。 这 参与转录的WDSV LTR的共识序列 将使用LTR缺失确定调节和反式激活 突变体。 该建议的主要目的是建立转录 使用mRNA的RT-PCR以及随后的克隆和 这些cDNA的测序。识别和表征 这些附件基因成绩单将为您提供必要的试剂 对这些基因及其编码的蛋白质进行研究 参与病毒基因表达。 这些实验应提供 进一步研究病毒附属作用的基础 肿瘤发生中的基因。格兰特= F32AI09301 逆转录病毒组装的机制在很大程度上未知。 尽管 在理解角色方面取得了进步 组装中GAG多蛋白前体的域几乎没有知道 关于该蛋白在此过程中如何与细胞相互作用。 此外，衣壳包膜的过程通常更差 理解。 原型D型的组装和萌芽 将研究逆转录病毒，梅森速效猴病毒。 该病毒 为拟议的研究提供了明显的优势，因为与 C型病毒，例如HIV，Capsid组装过程和 信封在细胞中在时间和空间上分离。 这 分离将允许检查这两个组装阶段 单独体外。 由于初步实验显示了 体外翻译用于组装研究的可行性这个系统将 被用来检查蛋白质和RNA的掺入 未成熟的衣壳。 M-PMV GAG突变体和HIV/M-PMV嵌合体的分析 将执行以划定有效效率所需的域 细胞质组件。 在体外和IN组装突变病毒 将比较体内以确定细胞的影响 该过程的运输机制。 另外， Capsid组件中的伴侣蛋白等细胞质成分将是 调查。 该系统也将用于分析 体外包膜。 同样，细胞成分的作用将 被调查。 逆转录病毒机理的详细描述 组装将促进开发的治疗抑制剂 复制。 此外，体外装配系统将提供 方便筛选此类潜在抑制剂的测定法。