Identification Novel Host Factors Regulating HIV Latency

静止记忆的CD4T细胞含有整合的、主动抑制的HIV前病毒，目前形成一种 根除病毒的巨大障碍。这种潜伏的蓄水池可以通过激活前病毒基因来攻击。 表达，从而使病毒对抗逆转录病毒治疗敏感。为了取得成功，这一战略必须既 防止病毒传播，导致所有潜伏感染的细胞死亡，而不会产生有毒状态 广泛性细胞激活。目前，还没有有效的治疗艾滋病毒潜伏期的方法。我们假设一个 对HIV潜伏期的分子基础有更全面的了解 促进和对抗潜伏期的宿主调节因素将促进发展 有效的治疗方法。在目标1中，我们将研究由基因组鉴定的五个新的候选艾滋病毒抑制因子 HIV感染的HeLa细胞的广泛siRNA筛选。这些候选生物具有一致的生物学特性 与潜伏期有关，并在淋巴组织中表达。这些候选基因的表达将是 在生物相关细胞中进行分析，并通过慢病毒shRNA敲除来评估它们的功能。在目标2中， 我们将在HIV潜伏期的CD4T细胞模型中筛选促进病毒潜伏期的microRNAs(MiR) 损害细胞激活物的表达。我们将在潜在的时间内使用对应器来验证MLR活动 被感染的原代CD4T细胞。在目标3中，我们将使用生物信息和转录分析方法来 鉴定被这些miRs抑制的宿主基因产物。使用这种双重实验方法， 我们将确定自然促进和拮抗HIV潜伏期的细胞因素。在适当情况下， 将进行作用机理研究。将根据稳健性确定确定的目标的优先顺序 用于小分子高通量筛选试验和小分子高通量筛选 将开发一次和二次筛查试验。这些拟议的研究可能会发现 抑制最感兴趣的细胞抑制物或激活最引人注目的mlRNA调节的分子 艾滋病毒激活剂。这些研究完全支持合作实验室确定组合体的目标 无毒的小分子可以根除病毒或产生功能性治愈(无药物缓解)。

Quiescent memory CD4 T cells harboring integrated, actively repressed HIV proviruses currently form a formidable barrier to viral eradication. This latent reservoir could be attacked by activating proviral gene expression thereby sensifizing the virus to anfiretroviral therapy . For success, this strategy must both prevent viral spread and result in the death of all latenfiy infected cells without producing a toxic state of generalized cellular activation. Currenfiy, no effective therapies for HIV latency exist. We hypothesize that a more complete understanding ofthe molecular underpinnings of HIV latency¿riotably, the full range of the host's regulatory factors that promote and antagonize latency¿will facilitate the development of effective therapies. In Aim 1, we will examine five novel candidate HIV repressors identified by genome wide siRNA screening of HIV-infected HeLa cells. These candidates have biological properties consistent with a role in latency and are expressed in lymphoid fissues. Expression of these candidate genes will be analyzed in biologically relevant cells and their function assessed by lentiviral shRNA knockdown. In Aim 2, we will screen a CD4 T-cell model of HIV latency for microRNAs (miRs) that promote viral latency by impairing the expression of cellular activators. We will validate mlR acfion using antagomirs in latently infected primary CD4 T-cells. In Aim 3, we will use bioinformafic and transcriptional profiling approaches to identify the host gene products that are suppressed by these miRs. Using this dual experimental approach, we will identify cellular factors that naturally promote and antagonize HIV latency. Where appropriate, mechanism-of-acfion studies will be performed. Idenfified targets will be prioritized, based on the robustness of their activity and overall "drugability," for entry into small-molecule high-throughput screening assays and primary and secondary screening assays will be developed. These proposed studies could identify small molecules that either inhibit the most interesfing cellular repressors or activate the most compelling mlRNAregulated HIV activators. These studies fully support the goal of the Collaboratory¿to identify combinafions of nontoxic small molecules that can eradicate the virus or produce a funcfional cure (drug-free remission).