SUPERANTIGEN FUNCTION IN MMTV INFECTION

描述(改编自申请人的摘要)：本报告的重点 建议是小鼠乳腺肿瘤的sag(超抗原)或orf基因 病毒(MMTV)。最近的实验表明，这种蛋白质，它是 编码在MMTV LTR中，在感染的小鼠中起超级抗原的作用。 这会导致TCR-β特异性T-β的整个子集缺失。 淋巴细胞。内源性MMTV前病毒的sag基因已被 发现与小鼠微量淋巴细胞刺激(MLS)相同 抗原。SAG基因在MMTV生命周期中的作用将是 在这些实验中进行了探索。在最近出版的著作中， 调查员和协查员表现出了一种 转基因小鼠体内外源性MMTV SAG使其对感染具有抵抗力 被相同的病毒感染，但不是其他MMTV。这意味着感染了T- 病毒传播途径中的淋巴细胞从肠道扩散到乳腺 腺体。最近的研究结果表明，SAG蛋白单独表达 延缓，但不能抑制转基因动物的肿瘤形成。在……里面 此外，来自SAG转基因动物的淋巴细胞不显示MLS 体外活性测定。另一方面，转基因动物 整个MMTV前病毒对肿瘤形成的抵抗力要强得多，而且 这些动物的淋巴细胞显示出MLS活性。这导致了 调查人员假设其他病毒基因(特别是env) 对于充分发挥超抗原功能是很重要的。一个全面的系列 提出了一系列实验，主要有四个目标。第一个目标 涉及SAG基因的分子操作。帧移位和 将在转基因小鼠身上测试SAG的缺失突变以确定 超抗原功能和阻断所必需的蛋白质结构域 病毒感染的可能性。此外，嵌合凹陷分子将是 产生以确定TCR V-β所涉及的蛋白质区域 特异性，SAG基因将被克隆和鉴定。第二 AIM将研究其亚细胞定位和组织分布 萨格。为了做到这一点，抗原性标记的SAG蛋白将 将开发针对SAG的特异性免疫试剂。 亚细胞定位将通过免疫沉淀进行研究，以及 组织分布将通过敏感的免疫学研究 免疫-聚合酶链式反应等技术。在第三个目标中，凹陷在 将对MMTV的生命周期进行调查。尤其是不同的淋巴组织 将对人群进行传染病病毒的检测，以及 它们是否能将病毒感染转移到乳腺细胞。这个 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 测试过。最终目的是研究MMTV包膜在sag基因中的作用 将研究功能和MLS响应。这将完成 通过在培养细胞或转基因小鼠中共表达env和sag，以及 测试env序列和sag序列是否可以在顺指令中协同工作 或者变性人。

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.