Revealing the HIV-1 Interactome

描述（由申请人提供）：鉴于HIV-1的遗传编码能力有限，可以合理地预期病毒必须与大量细胞因子相互作用才能完成其通过细胞的过程。的确，值得注意的是，病毒基因组仅占细胞全部遗传容量的0.0003%，却能利用细胞环境为己所用。然而，迄今为止，尽管HIV-1是研究最多的病毒，但只有相对较小的一组细胞蛋白被证明是HIV-1传播所必需的。一种遗传/蛋白质组学方法已经被开发出来，该方法利用随机诱变方案，遵循严格的生物选择步骤，努力查明HIV-1蛋白质组中那些罕见的位点，这些位点可以容纳一个有效的亲和力标签的结合，而不会显著丧失病毒复制能力所需的结构/功能关系。结合一种新的低温方法，在免疫分离期间捕获和保存短暂的病毒-宿主相互作用，我们已经恢复了几种与复制病毒相关的宿主蛋白，但以前在传统的科学研究中被掩盖了。我们确信我们的方法是合理的，因为在使用我们的方法回收和鉴定的细胞蛋白质中，有那些以前由其他小组确定与HIV-1相互作用的蛋白质。此外，提出了一种简单的质谱技术，可以高精度地区分免疫分离蛋白复合物中的特异性和非特异性蛋白质。所描述的结合遗传和蛋白质组学方法是一种强大的工具集，可用于识别在病毒生命周期中与给定病毒蛋白短暂关联的细胞蛋白，其中一部分可能是病毒生存所绝对需要的，但部分是宿主细胞所不需要的。公共卫生相关性：考虑到已知细胞内生物过程的复杂性以及HIV-1基因组小编码尺寸所施加的严格限制，我们有理由期望HIV-1蛋白质组必须依赖宿主因子阵列来完成其细胞内任务。为了努力恢复和识别与病毒机制相互作用的必要宿主蛋白，我们开发了一种系统的方法来选择可以编码小但有效的外来表位标签的衍生物，同时在培养中保持完全的复制能力。结合一种新的低温方法来捕获和保存短暂的病毒-宿主相互作用，我们已经恢复并鉴定了存在于病毒/宿主界面的新宿主蛋白组（与HIV-1相关，但以前在传统的科学研究中被掩盖），其中的一个子集可能有潜力为小分子干预这种病毒提供新的靶标。

DESCRIPTION (provided by applicant): Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive battery of cellular factors to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date and despite being easily the most studied virus, only a relatively small group of cellular proteins have been shown essential for HIV-1 propagation. A genetic/proteomic approach has been developed that utilizes a random mutagenic protocol followed by a stringent step of biological selection in efforts to pinpoint those rare sites within the HIV-1 proteome that can accommodate the incorporation of a potent affinity tag without significant loss of those structure/function relationships that are required for viral replication-competency. In conjunction with a novel cryogenic methodology to capture and preserve transient viral-host interactions during immunoisolation, we have recovered several host proteins in association with the replicating virus but previously obscured from conventional scientific investigation. We are reassured that our method is sound since among the cellular proteins recovered and identified using our methodologies are those previously determined by other groups to interact with HIV-1. Furthermore, a straightforward mass spectrometric technique is presented that discriminates with high accuracy between specific and nonspecific proteins in immunoisolated protein complexes. The combined genetic and proteomic method described is a powerful tool set that can be utilized to identify cellular proteins in transient association with a given viral protein at points during the viral life cycle, a subset of which may be absolutely required by the virus for its livelihood but in part, dispensable by the host cell. PUBLIC HEALTH RELEVANCE: Given the known complexity of biological processes within the cell as well as the rigid constraints imposed by the small coding size of the HIV-1 genome, it is reasonable to expect that the HIV-1 proteome must rely upon a battery of host factor arrays to complete its intracellular tasks. In an effort to recover and identify requisite host proteins that interact in complex with the viral machinery, we have developed a systematic method to select derivatives that can encode a small, but potent, foreign epitope tag yet remain fully replication-competent in culture. In conjunction with a novel cryogenic methodology to capture and preserve transient viral-host interactions, we have recovered and identified new sets of host proteins existing at the viral/host interface (in association with HIV-1 but previously obscured from conventional scientific investigation), a subset of which may have the potential to provide a new targets for small molecule intervention against this virus.