Revealing the HIV-1 Interactome

描述(申请人提供)：鉴于HIV-1的遗传编码能力有限，有理由预计该病毒必须与广泛的细胞因子相互作用才能完成细胞传递。事实上，值得注意的是，仅占细胞全部遗传能力约0.0003%的病毒基因组侵占了细胞环境，使之成为自己的优势。然而，到目前为止，尽管很容易成为研究最多的病毒，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。已经开发出一种遗传/蛋白质组学方法，该方法利用随机诱变方案，然后经过严格的生物选择步骤，努力定位HIV-1蛋白质组中那些能够容纳有效亲和标签的稀有位置，而不会显著损失病毒复制能力所需的那些结构/功能关系。结合一种新的低温方法来捕获和保存免疫分离过程中的瞬时病毒-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，但这些蛋白以前被传统的科学研究所掩盖。我们确信我们的方法是可靠的，因为在使用我们的方法恢复和鉴定的细胞蛋白中，有那些先前由其他小组确定的与HIV-1相互作用的蛋白。此外，还提出了一种简单的质谱学技术，该技术可以高精度地区分免疫分离蛋白复合体中的特定和非特定蛋白。所描述的遗传和蛋白质组相结合的方法是一套强大的工具，可用于在病毒生命周期的各个时间点识别与给定病毒蛋白瞬时相关的细胞蛋白，其中一部分可能是病毒赖以生存所必需的，但部分是宿主细胞所必需的。与公共卫生相关：鉴于细胞内生物过程的已知复杂性以及艾滋病毒-1基因组编码尺寸较小施加的严格限制，有理由预计艾滋病毒-1蛋白质组必须依赖一组宿主因子阵列来完成其细胞内任务。为了恢复和鉴定与病毒机制相互作用的必需宿主蛋白，我们开发了一种系统的方法来选择衍生物，这些衍生物可以编码一个小但有效的外源表位标签，但在培养中仍保持完全复制能力。结合一种新的低温方法来捕捉和保存瞬时的病毒-宿主相互作用，我们已经恢复和鉴定了存在于病毒/宿主界面的新的宿主蛋白集(与HIV-1相关，但以前被传统的科学研究所掩盖)，其中的一个子集可能有可能为针对这种病毒的小分子干预提供新的靶点。

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.