Are HIV-1-Infected Alveolar Macrophages Productive Sites of Viral Persistence?

项目总结/摘要 尽管有几个报告HIV-1感染的肺泡巨噬细胞（AM）在肺部的HIV-1感染， 在个体中，这些细胞在维持或持续感染中所起的作用仍然没有得到解决。 最近在马拉维进行的研究，对来自HIV-1感染者的AM进行了有效的病毒- 通过长期ART抑制，通过荧光原位检测可重复地检测HIV-1 mRNA的存在 杂交（FISH）。在这些研究中，我们还通过单细胞测序检测了HIV-1转录本 方案，并已分离出感染性HIV-1病毒从细胞回收支气管肺泡灌洗从艺术- 未感染HIV-1的志愿者。 我们提出的假设是，HIV-1转录本的存在，在肺泡巨噬细胞的艾滋病毒- 受感染个体的感染指示生产性病毒感染，其对于病毒的持续存在具有重要意义 在艺术。 R21阶段：目标1。HIV-1感染的AM是生产性感染吗？ 我们提出了共培养的方法来检测和捕获感染性HIV-1的AM和外周血， 马拉维的HIV-1感染志愿者。我们已经展示了使用允许的HIV-1易感的报告细胞， 我们可以从12名志愿者的试验性研究中从ART初治个体中获得传染性病毒。我们提出 将该分析扩展到更大的队列，包括ART治疗的病毒抑制个体。 R33阶段：目标2。HIV-1感染细胞中病毒和宿主转录物的转录谱。 利用马拉维已经建立的方法，我们将生成艾滋病毒感染者和 通过单细胞Seq-Well和Flow-FISH RNASeq方法， 单小区分辨率和覆盖深度。我们将使用这些数据集来探测HIV-1在细胞中的影响， 并研究来自AM衍生的HIV-1病毒的envs的细胞嗜性。 R33阶段：目标3。用合成mRNA和SiRNA干扰HIV-1感染的AM功能。 我们将使用功能获得（合成mRNA）和功能丧失（siRNA）的方法来操纵这些基因。 HIV-1感染的HMDM和AM的表型和行为。我们的目标是确定将推动 程序性细胞死亡，特别是在那些被HIV-1感染的AM中，作为选择性根除 这个潜在的病毒库 该提案基于这样的论点，即病毒抑制个体的AM将被证明是富有成效的- 感染了这一争论的验证是这一阶段性合同R21阶段的重要里程碑。

Project Summary / Abstract Despite several reports of HIV-1-infected alveolar macrophages (AM) in the lungs of HIV-1-infected individuals, the roles played by these cells in the maintenance or persistence of infection remain unresolved. Recent studies in Malawi, conducted on AM from HIV-1-infected individuals that are effectively virally- suppressed by long-term ART, reproducibly detected the presence of HIV-1 mRNA by fluorescent in situ hybridization (FISH). In these studies, we also detected HIV-1 transcripts through single cell sequencing protocols, and have isolated infectious HIV-1 virus from cells recovered by bronchoalveolar lavage from ART- naïve, HIV-1-infected volunteers. The hypothesis we propose to test is that the presence of HIV-1 transcripts in the alveolar macrophages of HIV- infected individuals is indicative of a productive viral infection that has significance for persistence of the virus during ART. R21 Phase: Aim 1. Are HIV-1-infected AM productively infected? We propose co-culture approaches to detect and capture infectious HIV-1 from the AM and peripheral blood of HIV-1-infected volunteers in Malawi. Using permissive, HIV-1-susceptible reporter cells we have already shown that we can acquire infectious virus from ART-naïve individuals in a pilot study on 12 volunteers. We propose expanding this analysis to a larger cohort, including ART-treated, virally-suppressed individuals. R33 Phase: Aim 2. Transcriptional Profiling of Viral and Host Transcripts in HIV-1-Infected Cells. Using methods already established in Malawi, we will generate transcriptional profiles of HIV-infected and uninfected AMs by single-cell Seq-Well and Flow-FISH RNASeq methods to obtain datasets reflecting both single-cell resolution and depth of coverage. We will use these datasets to probe the impact of HIV-1 in cell longevity and to study the cellular tropism of the envs from AM-derived HIV-1 virus. R33 Phase: Aim 3. Perturbation of HIV-1-infected AM Function with Synthetic mRNA and SiRNA. We will use gain-of-function (synthetic mRNA) and loss-of-function (siRNA) approaches to manipulate the phenotype and behavior of HIV-1-infected HMDMs and AMs. The goal is to identify pathways that will drive programmed cell death specifically in those AMs that are HIV-1-infected as a route for selective eradication of this potential viral reservoir. The proposal is based on the contention that AM in virally-suppressed individuals will prove to be productively- infected. The verification of this contention is the major milestone of the R21 period of this phased award.