RETROVIRUS REGULATION IN VITRO AND DURING DEVELOPMENT

本方案中试验的目标是确定结构 与禽白血病-肉瘤病毒的功能关系 禽类内源性LTRS中的增强子和增强子序列 病毒(EVS)。非急性转化禽白血病病毒(ALV) 感染某些病毒后会导致法氏囊淋巴瘤的高发 敏感品系的鸡。通过这些进行细胞转化 病毒通常与前病毒DNA在或附近整合有关 在细胞癌基因c-myc中，导致表达增加 该基因在启动子/增强子序列的控制下 前驱LTR的U3区。相比之下，禽类内源性病毒 (EV)在体内具有弱致瘤性。有人建议说，低水平 禽类EVS的致瘤性是应有的 主要是由于他们的ltrs中没有强大的增强剂来防止 有效地调节顺式激活事件，如所看到的 在外源病毒诱导的肿瘤中。 然而，出乎意料的是，在EV LTRs中发现了 能够取代，在一个方向独立的水手，基本 与它们共享的外源病毒LTR中的增强子序列 有限的序列相似性。构建EV-外源病毒杂交体 该效果所需的EV LTRS内的序列正在 本地化。还获得了初步数据，表明EV 增强子序列与细胞蛋白质相互作用 外源病毒增强子结合蛋白。因此，增强子基序在 EV Ltr与外源病毒Ltr有明显差异。这个 高效转录的杂交LTRs的可用性 来自弱致癌EV的增强子基序提供了机会 直接测试增强子活性、身份之间的关系 和不同的增强子基序的功能，以及肿瘤的发生。这将是 使用蛋白质结合研究和 单个EV和外源病毒增强子域的功能分析 EV-外源病毒杂交种LtrS。然后，选定的混合LTR将 用于生成具有复制能力的病毒，以确定其 体外生长速度，并产生病毒库存，供未来使用 致癌性检测。这些研究的长期目标是确定 并潜在地区分调解 在体外实验中增强启动子活性，在未来，那些 是体内高水平的肿瘤发生所必需的。

The goal of experiments in this proposal is to determine the structural and functional relationship between the avian leukosis-sarcoma virus enhancer and enhancer sequences within the LTRs of avian endogenous viruses (evs). Non-acute transforming avian leukosis viruses (ALVs) induce a high incidence of bursal lymphomas after infection of certain susceptible strains of chickens. Cellular transformation by these viruses is commonly associated with integration of proviral DNA near or within the cellular oncogene c-myc which results in increased expression of this gene under the control of promoter/enhancer sequences within the U3 region of the proviral LTR. In contrast, the avian endogenous viruses (evs) are weakly oncogenic in vivo. It has been suggested that low level oncogenicity of avian evs is due primarily to the absence of a strong enhancer in their LTRs that prevents them from efficiently mediating cis-activation events such as those seen in exogenous virus-induced tumors. Unexpectedly however, sequences have been identified in ev LTRs that are able to replace, in an orientation independent mariner, essential enhancer sequences in the exogenous virus LTR with which they share limited sequence similarity. By constructing ev-exogenous virus hybrid LTRs, the sequences within ev LTRs required for this effect are being localized. Preliminary data has also been obtained indicating that ev enhancer sequences interact with cellular proteins distinct from exogenous virus enhancer binding proteins. Thus, enhancer motifs within the ev LTR appear distinct from those in the exogenous virus LTR. The availability of hybrid LTRs that are transcribed efficiently yet contain enhancer motifs from the weakly oncogenic ev provides the opportunity to directly test the relationships between enhancer activity, the identity and function of distinct enhancer motifs, and oncogenesis. This will be investigated using a combined approach of protein binding studies and functional analyses on individual ev and exogenous virus enhancer domains and on ev-exogenous virus hybrid LTRs. Selected hybrid LTRs will then be used to generate replication competent virus in order to determine their growth rates in vitro and to generate virus stocks for future use in oncogenicity testing. The long range goal of these studies is to define and to potentially distinguish between regulatory elements that mediate enhancer activity in in vitro experiments and, in the future, those that are required for high level oncogenesis in vivo.