Revealing the HIV-1 Interactome

揭示 HIV-1 相互作用组

• 批准号: 7685768
• 负责人: MARK AYER MUESING
• 金额: $ 45.63万
• 依托单位: AARON DIAMOND AIDS RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7685768
• 关键词: Affinity; Antigens; Antiviral Agents; Binding; Biochemical; Biological; Biological Preservation; Biological Process; Cell physiology; Cells; Classification; Code; Complex; DNA; DNA Viruses; Engineering; Environment; Enzymes; Epitopes; Funding; Gene Targeting; Genetic; Genetic Code; Genome; Growth; HIV; HIV Infections; HIV-1; HIV-1 integrase; Human; Human Activities; Infection; Integrase; Integration Host Factors; Intervention; Investigation; Life Cycle Stages; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Mutagenesis; Nuclear Import; Procedures; Process; Proteins; Proteome; Proteomics; Protocols documentation; Reverse Transcription; Site; Site-Directed Mutagenesis; Source; Structure-Activity Relationship; Surface; Surveys; System; T-Lymphocyte; Techniques; Ubiquitination; Vaccines; Viral; Viral Envelope Proteins; Viral Genome; Viral Proteins; Virion; Virus; base; cryogenics; env Gene Products; immunological intervention; insight; novel; protein complex; protein protein interaction; public health relevance; recombinant virus; small molecule; sound; tool; vif Gene Products; viral DNA

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.

描述（由申请人提供）：鉴于HIV-1的基因编码能力有限，可以合理地期望该病毒必须与大量的细胞因子相互作用才能完成其通过细胞的通过。确实，值得注意的是，病毒基因组仅占细胞整个遗传能力的0.0003％，指挥官具有自身的优势。然而，迄今为止，尽管很容易成为研究最多的病毒，但仅显示了一组相对较小的细胞蛋白对于HIV-1传播至关重要。已经开发了一种遗传/蛋白质组学方法，该方法利用了随机的诱变方案，然后采取了严格的生物选择步骤，以确定HIV-1蛋白质组中的稀有位点，这些位点可以适应有效的亲和力标签，而无需严重损失那些结构/功能关系，而这些结构/功能关系是病毒复制能力所必需的。结合一种新型的低温方法，可以在免疫分离过程中捕获和保留短暂的病毒宿主相互作用，我们已经与复制病毒相关的几种宿主蛋白​​回收了几种宿主蛋白​​，但以前从常规的科学研究中掩盖了。我们放心的是，我们的方法是正确的，因为在使用我们的方法恢复和鉴定的细胞蛋白中，其他组先前由其他组确定的方法与HIV-1相互作用。此外，提出了一种直接的质谱技术，该技术在免疫溶解的蛋白质复合物中特异性和非特异性蛋白之间的歧视较高。所描述的遗传和蛋白质组学方法组合是一个强大的工具集，可以用来鉴定病毒生命周期中与给定病毒蛋白在瞬时关联的细胞蛋白质，该病毒生命的子集可能绝对需要其生计，但部分地是由宿主细胞dopensensent。公共卫生相关性：鉴于细胞内生物过程的已知复杂性以及HIV-1基因组的较小编码大小所施加的刚性约束，因此可以合理地期望HIV-1蛋白质组必须依靠一系列宿主因子阵列来完成其细胞内任务。为了恢复和识别与病毒机械复杂相互作用的必要的宿主蛋白，我们开发了一种系统的方法来选择可以编码一个小但有效的外位表标签但在培养中仍然完全复制的衍生物。结合一种新型的低温方法来捕获和保留瞬态病毒宿主相互作用，我们已经恢复并确定了现有在病毒/宿主界面处存在的新的宿主蛋白集（与HIV-1相关联，但以前从常规科学研究中掩盖了），其中一个子集可能有可能为这种小分子介入提供新的靶标。