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

项目摘要/摘要 尽管有几份关于HIV-1感染患者肺内肺泡巨噬细胞(AM)的报道 对于个体而言，这些细胞在维持或持续感染方面所起的作用仍未解决。 最近在马拉维进行的研究，对来自HIV-1感染者的AM进行了研究，这些人实际上是病毒性的- 经长期ART抑制，用原位荧光技术重复性检测到HIV-1mRNA的存在 杂交(鱼)。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，并已从ART-ART经支气管肺泡灌洗回收的细胞中分离出传染性HIV-1病毒。 天真的、感染了HIV-1的志愿者。 我们提出的假设是，HIV-1转录本在HIV-1的肺泡巨噬细胞中的存在- 被感染的个体表明有生产力的病毒感染，这对病毒的持久性具有重要意义。 在艺术上。 R21阶段：目标1.感染HIV-1的AM是否有效感染？ 我们提出了从AM和外周血中检测和捕获传染性HIV-1的共培养方法 马拉维感染HIV-1的志愿者。使用我们已经展示过的允许的、对HIV-1敏感的报告细胞 在一项对12名志愿者进行的初步研究中，我们可以从天真的ART患者身上感染到传染性病毒。我们建议 将这一分析扩大到更大的队列，包括接受抗逆转录病毒治疗、病毒抑制的个人。 R33阶段：目标2。HIV-1感染细胞中病毒和宿主转录产物的转录图谱。 使用马拉维已经建立的方法，我们将生成艾滋病毒感染者和 用单细胞序列井和Flow-FISH RNAseq方法获得反映两者的数据集的未感染AM 单元格分辨率和覆盖深度。我们将使用这些数据集来探索HIV-1在细胞中的影响 目的：研究AM来源的HIV-1病毒包膜的细胞亲和性。 R33阶段：目的3.合成的mRNA和siRNA对HIV-1感染的AM功能的干扰。 我们将使用功能增益(合成mRNA)和功能损失(SiRNA)方法来操纵 HIV-1感染的HMDM和AM的表型和行为。目标是确定将驾驶的路径 程序性细胞死亡，特别是在那些感染HIV-1的AM中，作为选择性根除 这个潜在的病毒库。 这项建议是基于这样一种观点，即病毒抑制的个体的AM将被证明是有效的- 被感染了。这一争辩的验证是这一阶段性奖项R21期间的重大里程碑。

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.