Oncogenesis By Jaagsiekte Sheep Retrovirus

描述（由申请人提供）：我们将继续研究jaagsiekte绵羊逆转录病毒（JSRV）的致癌转化，JSRV是一种传染性肺癌，绵羊肺癌（OPA）的病因，与人类肺腺癌非常相似。肿瘤发生的靶细胞是II型肺细胞和Clara细胞。JSRV的独特之处在于它的包膜基因也是一个致癌基因；它可以转化培养细胞，在动物体内诱导肿瘤。像其他病毒癌蛋白一样，Env的多个结构域参与了转化。跨膜蛋白（TM）的胞质尾部是必不可少的，但表面蛋白（SU）也参与其中。此外，JSRV编码一种调控活性rej，这是非剪接病毒mRNA蛋白表达所必需的。Rej在env区域内编码，这增加了它也参与转换的可能性。本研究主要有三个目的：1)对Env蛋白TM和rej的结构功能进行分析。通过溶液核磁共振测定TM的细胞质尾部结构，研究TM的跨膜结构域和外结构域的潜在作用，并评估rej在转化中的潜在作用。2)鉴定参与转化的细胞蛋白。通过酵母双杂交筛选确定的候选蛋白将测试与JSRV转化的相关性，并通过串联亲和纯化(TAP)/蛋白质组学方法寻找额外的细胞结合蛋白。3)体外和体内JSRV转化。为了更密切地反映肺上皮细胞的体内肿瘤发生，还将测试大鼠和羊II型肺细胞（带或不带协同致癌基因）以及小鼠细支气管-肺泡干细胞（BASCs）的体外转化。研究人员将建立JSRV诱导肺癌的转基因小鼠模型，并在腺相关病毒（AAV）载体上测试野生型和突变型JSRV Env的致瘤性。这些实验将大大增加我们对这种独特的人类肺癌模型的理解，并且发现的致癌途径可能阐明人类肺癌的类似途径。

DESCRIPTION (provided by applicant): We will continue studies on oncogenic transformation by jaagsiekte sheep retrovirus (JSRV), the cause of a transmissible lung cancer, ovine pulmonary carcinoma (OPA) that closely resembles human lung adenocarcinoma. The target cells for oncogenesis are type II pneumocytes and Clara cells. JSRV is unique in that its envelope gene also is an oncogene; it can transform cells in culture and induce tumors in animals. Like other viral onco-proteins, multiple domains of Env are involved in transformation. The cytoplasmic tail of the transmembrane protein (TM) is essential, but the surface protein (SU) also participates. In addition, JSRV encodes a regulatory activity rej, necessary for expression of proteins from unspliced viral mRNA. Rej is encoded within the env region, which raises the possibility that it may also be involved in transformation. The proposed research has three aims: 1) Structure-function analysis of the Env protein TM, and of rej. The structure of the cytoplasmic tail of TM will be determined by solution NMR, the potential roles of the membrane-spanning and ectodomains of TM will be investigated, and a potential role for rej in transformation will be evaluated. 2) Identification of cellular proteins involved in transformation. Candidate proteins identified by yeast two-hybrid screening will be tested for relevance to JSRV transformation, and additional cellular binding proteins will be sought by tandem affinity purification (TAP)/proteomic approaches. 3) JSRV transformation in vitro and in vivo. To more closely mirror in vivo oncogenesis of lung epithelial cells, in vitro transformation of primary rat and ovine type II pneumocytes (with or without cooperating oncogenes), and of murine bronchiole-alveolar stem cells (BASCs) will also be tested. A transgenic mouse model for JSRV-induced lung cancer will be developed, and wild-type and mutant JSRV Env's will be tested for tumorigenicity in an adeno-associated virus (AAV) vector. These experiments will substantially increase our understanding of this unique model for human lung cancer, and the oncogenic pathways uncovered may elucidate similar ones in human lung cancer. Lay summary: This proposal will study a virus of sheep (JSRV) that causes a transmissible lung cancer (OPA), very similar to one form of human lung cancer. The discoveries may lead to a better understanding of human lung cancer, which may result in improved therapies and prevention.