The Genitourinary Tract as a compartment and reservoir for HIV

 描述(申请人提供)：尽管联合抗逆转录病毒疗法显著改善了艾滋病毒相关的发病率和死亡率，但仍然存在一些挑战，包括多个潜伏的病毒储备库能够在 艺术的缺失。作为治疗计划的一部分，根除这些宿主的任何努力都需要了解艾滋病毒重新激活和在长期携带病毒的组织和细胞中复制的动态和控制。这项拟议的研究分析了生殖道(GU)作为艾滋病毒的独特隔间和储存库。我们以前的工作已经证明肾上皮细胞是一个独特的病毒区段，在初步数据中，来自男性和女性尿液的病毒RNA序列形成了独立的簇(S)，与来自外周血单核细胞和血浆的病毒序列形成了不同的簇，这与病毒在胃肠道中的区段划分一致。尿液中的病毒检测允许对这个隔间进行重复采样，这在病毒反弹研究中特别有用。为了确定男性尿液衍生序列的来源，将同时从尿液和精液中分离出无细胞和细胞相关的病毒序列，并对其进行基因特征分析。为了评估胃肠道对病毒反弹的贡献，将对参加ACTG 5345试验(特定目标1)的患者在抗逆转录病毒治疗期间和治疗中断后，对尿液来源的艾滋病毒核酸进行同步和纵向系统发育序列分析，并与外周血单个核细胞和血浆进行比较。为了评估HIV核酸在上消化道的长期体内持久性，将对长期抑制ART的HIV阳性患者的肾活检组织进行DNA和RNA扩增、原位杂交和深度测序。深度测序将检测与受感染的肾上皮细胞克隆性扩张相关的优先HIV整合位点(特定目标2)。具体目标3将定义肾脏的长期潜力 作为艾滋病毒的潜在和可重新激活的蓄水池。一种双重荧光病毒结构，允许鉴定和纯化含有转录活性或沉默的细胞群体 HIV，将被用来确定病毒重新激活的生理和药物策略(从潜伏期开始)以及RTE在激活时的命运。在体外方法中，从被抑制的HIV-1阳性受试者的尿液中分离和培养的RTE细胞将使用类似的激活策略进行评估，并与同时获得的PBMC的刺激进行比较。总的来说，这项工作将进一步描述gu道相关病毒的特征，并确定其特征。 肾上皮细胞的潜伏期和转录激活的改变，这是一个独特的病毒隔间，可能有助于艾滋病毒的长期存在。

 DESCRIPTION (provided by applicant): Despite the dramatic improvement in HIV-associated morbidity and mortality from combination antiretroviral therapy, a number of challenges remains including the long-term persistence of multiple, latent viral reservoirs capable of reactivation in the absence of ART. Any effort to eradicate these reservoirs as part of a cure initiative requires understanding of the dynamics and control of HIV reactivation and replication in tissues and cells harboring the virus long-term. The proposed studies analyze the genitourinary (GU) tract as a unique compartment and reservoir for HIV. Our prior work has demonstrated that renal epithelial cells are a unique viral compartment and in preliminary data we show that viral RNA sequences derived from the urine of males and females form separate cluster(s) from those derived from PBMC and plasma, consistent with viral compartmentalization in the GU tract. Viral detection in the urine allows for repeated sampling of this compartment, which can be particularly useful in viral rebound studies. To determine the source of urine- derived sequences in males, cell free and cell-associated viral sequences will be isolated and genetically characterized simultaneously from urine and semen. To assess the contribution of the GU tract to virus rebound, simultaneous and longitudinal phylogenetic sequence analysis will be performed on urine-derived HIV nucleic acids in comparison with PBMC and plasma, during ART and after treatment interruption in patients enrolled in the ACTG 5345 trial (Specific Aim 1). To assess the long-term in vivo persistence of HIV nucleic acids in the upper GU tract, DNA and RNA amplification, in situ hybridization and deep sequencing will be performed on kidney biopsies from HIV positive subjects that have prolonged suppression on ART. Deep sequencing will detect preferential HIV integration sites associated with clonal expansion of infected renal epithelial cells (Specific Aim 2). Specific Aim 3 will define the long-term potential of the kidney as a latent and reactivatable reservoir for HIV. A dual fluorescent viral construct, that allows identification and purification of cell populations containing transcriptionally active or silenced HIV, will be used to define physiologic and pharmacologic strategies for virus reactivation (from latency) as well as the fate of RTE upon activation. In an ex vivo approach, RTE cells isolated and cultured from the urine of suppressed HIV-1 positive subjects will be assessed using similar activation strategies and compared to stimulation of simultaneously derived PBMC. Collectively, this work will further characterize the GU tract associated virus as well as define characteristics of latency and transcriptional activation in renal epithelium, a unique viral compartment that may contribute to long-term HIV persistence.