Proteomics and phosphoproteomics analysis in HIV-1 infection

摘要 感染HIV-1后，免疫系统会发生复杂的、微调的免疫反应，几乎涉及免疫系统的所有组成部分。 先天性和适应性免疫系统。然而，这种免疫反应在大多数情况下无法控制感染， 有理由相信，在许多情况下，对艾滋病毒的免疫反应有助于疾病的发病机制 通过增加异常免疫激活和促进建立HIV-1的持久储存库 感染的细胞，从而传播疾病，他们是为了限制。而先天性和适应性效应细胞 在过去的几年里，对HIV-1的反应进行了详细的分析，仍然有一个显着的知识 在理解决定CD 4 T细胞内的分子事件和宿主-病原体相互作用方面存在差距 HIV-1生命周期的功效，并影响HIV-1感染细胞的命运和存活。然而，免疫 在实际的病毒靶细胞内对HIV-1的反应可以说是最活跃的反应之一， 最有效的抗病毒免疫防御机制，可能对临床动态产生深远的影响。 HIV-1疾病进展。在这里，我们建议利用最近的方法进步，为高- 通量、超灵敏蛋白和磷蛋白谱分析，以分析分子事件和细胞内在 HIV-1的主要靶细胞内对HIV-1的免疫反应。在具体目标1中，我们将重点关注 HIV-1基因产物被宿主激酶磷酸化。我们假设， HIV-1蛋白质组在体内易被宿主激酶磷酸化，并代表 对病毒适应性有深远影响的非凡病毒脆弱性;识别此类网站可能具有 重要的影响，以产生一个全面的看法，艾滋病毒-1的健身景观和选择病毒 包括在疫苗和免疫原中的序列。在具体目标2中，我们将分析蛋白质组学和 磷酸化蛋白质组反应生产性HIV-1感染，并测试假设，HIV-1感染诱导 维持受感染细胞的存活和稳态的特定分子途径。这些研究建立在 大量的挑衅性初步数据表明，HIV-1感染细胞的蛋白质组特征显示， 选择性激活细胞存活程序，并有望确定减少 病毒感染细胞的存活力和持久性。在具体目标3中，我们将重点分析蛋白质组学， 潜在感染的CD 4 T细胞的磷酸化蛋白质组学特征，这被认为是我们研究的主要原因。 目前在根除和治愈HIV-1感染方面还存在不足。这些研究有可能发现新的 参与调节病毒潜伏期和持久性的功能途径，并将提供数据， 可以显著增强我们检测、监测和治疗性消除潜在感染细胞的能力。

Abstract Infection with HIV-1 elicits complex, fine-tuned immune responses that involve virtually all components of the innate and adaptive immune system. Yet, this immune response fails in most cases to control the infection, and there are reasons to believe that in many cases, immune responses to HIV contribute to disease pathogenesis by increasing abnormal immune activation and facilitating the establishment of a long-lasting reservoir of HIV-1 infected cells, thus propagating a disease they are meant to restrict. While innate and adaptive effector cell responses against HIV-1 have been analyzed in detail in the last years, there is still a remarkable knowledge gap in the understanding of molecular events and host-pathogen interactions inside CD4 T cells that determine the efficacy of the HIV-1 life cycle and influence the fate and survival of HIV-1 infected cells. However, immune responses to HIV-1 within the actual viral target cells arguably represent one of the most active and one of the most effective antiviral immune defense mechanisms that may have a profound effect on the dynamics of clinical HIV-1 disease progression. Here, we propose to take advantage of recent methodological advances for high- throughput, ultra-sensitive protein and phosphoprotein profiling to analyze molecular events and cell-intrinsic immune responses to HIV-1 inside the main target cells for HIV-1. In Specific Aim 1, we will focus on phosphorylation of HIV-1 gene products by host kinases. We hypothesize that specific sequence motifs within the HIV-1 proteome are susceptible to phosphorylation by host kinases in vivo, and represent sites of extraordinary viral vulnerability that have a profound impact on viral fitness; identifying such sites may have important implications for generating a comprehensive view of the HIV-1 fitness landscape and for selecting viral sequences to be included in vaccines and immunogens. In Specific Aim 2, we will analyze proteomic and phosphoproteomic responses to productive HIV-1 infection, and test the hypothesis that HIV-1 infection induces specific molecular pathways that maintain survival and homeostasis of infected cells. These studies build on a substantial set of provocative preliminary data indicating that proteomic signatures of HIV-1 infected cells show selective activation of cell survival programs, and hold promise for identifying molecular targets for reducing viability and persistence of virally infected cells. In Specific Aim 3, we will focus on analyzing the proteomic and phosphoproteomic signatures of latently-infected CD4 T cells, which are regarded as the main reason for our current insufficiency to eradicate and cure HIV-1 infection. These studies have the potential to discover novel functional pathways that are involved in regulation of viral latency and persistence, and will provide data that may significantly enhance our ability to detect, monitor and therapeutically eliminate latently-infected cells.