SUPERANTIGEN FUNCTION IN MMTV INFECTION

MMTV 感染中的超抗原功能

• 批准号: 2429731
• 负责人: Jaquelin Page Dudley
• 金额: $ 35.48万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2429731
• 关键词: T cell receptor; frameshift mutation; gene deletion mutation; genetic strain; genetically modified animals; immunoprecipitation; laboratory mouse; life cycle; mammary gland; molecular cloning; molecular genetics; mouse mammary tumor virus; neoplastic cell; nucleic acid sequence; polymerase chain reaction; protein structure function; superantigens; tissue /cell culture; transfection /expression vector; viral carcinogenesis; virus antigen; virus envelope; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (Adapted from the applicant's abstract): The focus of this proposal is the Sag (super-antigen) or orf gene of murine mammary tumor virus (MMTV). Recent experiments indicate that this protein, which is encoded in the MMTV LTR, functions as a super-antigen in infected mice. This leads to deletion of whole subsets of TCR-beta-specific T- lymphocytes. The Sag genes of endogenous MMTV proviruses have been found to be the same as the murine minor lymphocyte stimulating (Mls) antigens. The role of the Sag gene in the MMTV life cycle will be explored in these experiments. In recently published work, the investigator and co-investigator have shown that expression of an exogenous MMTV Sag in transgenic mice renders them resistant to infection by the same virus but not other MMTVs. This implicates infection of T- lymphocytes in the pathway of virus spread from the gut to the mammary gland. Recent results indicate that expression of Sag protein alone delays, but does not inhibit tumorigenesis in transgenic animals. In addition, lymphocytes from Sag transgenic animals do not display Mls activity in in vitro assays. On the other hand, animals transgenic for an entire MMTV provirus are much more resistant to tumorigenesis, and lymphocytes from these animals show Mls activity. This leads the investigators to hypothesize that other viral genes, (env in particular) are important for full superantigen function. A comprehensive series of experiments are proposed, with four major aims. The first aim involves molecular manipulations of the Sag gene. Frame shift and deletion mutations of Sag will be tested in transgenic mice to identify domains of the protein necessary for superantigen function and blockage of viral infection. In addition, chimeric Sag molecules will be generated to determine the region of the protein involved in TCR V-beta specificity, and Sag mRNA will be cloned and characterized. The second aim will study the subcellular localization and tissue distribution of Sag. In order to do this, antigenically tagged Sag protein will be generated, and Sag-specific immunological reagents will be developed. The subcellular localization will be studied by immunoprecipitation, and tissue distribution will be studied by sensitive immunological techniques such as immuno-PCR. In the third aim, the role of Sag in the MMTV life cycle will be investigated. In particular, different lymphoid populations will be tested for the presence of infectious virus, and whether they can transfer viral infection to mammary gland cells. The effect of Sag gene mutations on viral infectivity will be tested, and infection in mutant mice lacking different lymphoid components will be tested. In the final aim, the role of MMTV envelope in Sag gene function and Mls response will be investigated.This will be accomplished by co-expressing env and Sag in cultured cells or transgenic mice, and testing whether the env sequences and sag sequences can cooperate in cis or trans.

描述（改编自申请人的摘要）： 提案是鼠乳腺肿瘤的SAG（超抗原）或ORF基因 病毒（MMTV）。 最近的实验表明该蛋白质，即 在MMTV LTR中编码，在感染小鼠中充当超抗原。 这导致删除TCR-beta特异性T-的整个亚集 淋巴细胞。 内源性MMTV病毒的SAG基因一直是 发现与鼠小淋巴细胞刺激（MLS）相同 抗原。 SAG基因在MMTV生命周期中的作用将是 在这些实验中探讨了。 在最近发表的工作中 研究者和共同评估者表明 转基因小鼠中的外源MMTV下垂使它们对感染有抵抗力 通过同一病毒，而不是其他MMTV。 这意味着T-感染 病毒途径中的淋巴细胞从肠道传播到乳腺 腺。 最近的结果表明，单独表达SAG蛋白 延迟，但不抑制转基因动物的肿瘤发生。 在 此外，来自SAG转基因动物的淋巴细胞不显示ML 体外测定中的活性。 另一方面，动物转基因 整个MMTV病毒对肿瘤的耐药性更大，并且 这些动物的淋巴细胞显示MLS活性。 这导致 研究人员假设其他病毒基因（特别是ENV） 对于完整的超抗原功能很重要。 一个全面的系列 提出了实验的四个主要目标。 第一个目标 涉及SAG基因的分子操作。 框架移动和 SAG的缺失突变将在转基因小鼠中进行测试，以识别 超抗原功能和阻塞所需的蛋白质域 病毒感染。 另外，嵌合SAG分子将是 生成以确定参与TCR V-beta的蛋白质区域 特异性和SAG mRNA将被克隆和表征。 第二个 AIM将研究细胞的本地化和组织分布 下垂。 为此，抗原标记的SAG蛋白将是 将开发出生成的，SAG特异性免疫试剂。 亚细胞定位将通过免疫沉淀研究，并且 组织分布将通过灵敏的免疫学研究 诸如免疫PCR之类的技术。 在第三个目标中，SAG在 将研究MMTV生命周期。 特别是不同的淋巴样 种群将被测试是否存在传染病，并 它们是否可以将病毒感染转移到乳腺细胞中。 这 将测试SAG基因突变对病毒感染性的影响，并 缺乏不同淋巴成分的突变小鼠的感染将是 测试。 在最终目标中，MMTV信封在SAG基因中的作用 将研究功能和MLS响应。 通过在培养的细胞或转基因小鼠中共表达ENV和下垂，以及 测试ENV序列和SAG序列是否可以在CIS中配合 或trans。