SUPERANTIGEN FUNCTION IN MMTV INFECTION

描述（改编自申请人的摘要）：本文的重点 本发明提出了一种小鼠乳腺肿瘤的Sag（超抗原）或orf基因， MMTV病毒。 最近的实验表明，这种蛋白质， 在MMTV LTR中编码，在感染的小鼠中作为超抗原发挥作用。 这导致TCR-β特异性T细胞亚群的全部缺失。 淋巴细胞 内源性MMTV前病毒的Sag基因已经被研究。 发现与小鼠次要淋巴细胞刺激（Mls）相同 抗原 Sag基因在MMTV生命周期中的作用将是 在这些实验中探索。 在最近出版的作品中， 研究者和共同研究者已经表明， 转基因小鼠中的外源性MMTV Sag使它们对感染具有抗性 但不是其他的MMTV 这意味着T- 淋巴细胞在病毒从肠道传播到乳腺的途径中 腺。 最近的结果表明，Sag蛋白的表达单独 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Mls， 在体外测定中的活性。 另一方面，转基因动物 完整的MMTV前病毒对肿瘤发生的抵抗力更强， 来自这些动物的淋巴细胞显示Mls活性。 这导致了 研究人员推测，其他病毒基因，（特别是env） 对超抗原的完整功能很重要 一系列综合 实验提出了四个主要目标。 第一个目标 涉及对Sag基因的分子操作。 帧移位和 将在转基因小鼠中测试Sag的缺失突变以鉴定 超抗原功能和阻断所必需的蛋白质结构域 病毒感染。 此外，嵌合Sag分子将被 产生以确定涉及TCR V-β的蛋白质的区域 特异性，并且将克隆和表征Sag mRNA。 第二 目的是研究亚细胞定位和组织分布， 凹陷。 为了做到这一点，抗原标记的Sag蛋白将被 产生，并将开发Sag特异性免疫试剂。 将通过免疫沉淀研究亚细胞定位， 将通过敏感的免疫学方法研究组织分布。 免疫PCR等技术。 在第三个目标中，凹陷在 MMTV的生命周期将被调查。 特别是，不同的淋巴 将检测人群中是否存在传染性病毒， 它们是否能将病毒感染转移到乳腺细胞。 的 将测试Sag基因突变对病毒感染性的影响， 缺乏不同淋巴成分的突变小鼠的感染将是 测试. 在此基础上，研究了MMTV囊膜蛋白在Sag基因中的作用， 功能和Mls响应将被调查。这将被完成 通过在培养的细胞或转基因小鼠中共表达env和Sag，和 测试env序列和sag序列是否可以在cis中协作 或反式

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.