Molecular profile of proviral reservoirs in HIV-infected drug users

摘要 摘要 使用注射毒品的人是感染HIV-1的最高风险人群，他们的亲属 在全世界范围内，艾滋病毒1型感染者的总人数正在增加。这一点对于 美国约有200万人受到阿片类药物危机的影响，其中约9%（18万人） 目前估计是HIV-1感染者。由于最近在改善获得护理和 坚持治疗，这些人中有相当一部分现在能够维持不可检测的病毒 持续使用阿片类药物（羟考酮、海洛因、氢吗啡酮、芬太尼）或阿片类替代药物期间的负荷 （美沙酮或丁丙诺啡）。然而，病毒感染细胞的残余储库在这些患者中持续存在， 并且代表了对抗病毒感染的长期无药物缓解的主要屏障。最近有 在理解病毒储存库持久性的细胞区室和机制方面取得了实质性进展， 但阿片类药物成瘾者要么人数严重不足，要么完全被排除在此类调查之外。然而， 有理由相信阿片类药物使用者中病毒库的大小，结构和组成基本上是 与不使用药物的HIV-1感染者不同。例如，阿片类药物滥用可以深刻地 改变基因表达模式，并诱导表观遗传染色质修饰，这两者都是已知的 影响逆转录病毒感染的易感性，染色体整合位点的选择和 整合的HIV-1前病毒的转录活性。本项目着手对 HIV-1感染的阿片类药物成瘾者中的病毒储库结构和组成，使用一系列新的下一代- 一代测序技术，允许在前所未有的广度上分析病毒库 和深度在具体目标1中，我们将全面分析完整的HIV-1的染色体定位 前病毒，基于一种新的实验方法，结合全基因组扩增，近全长病毒 测序和连接介导的PCR用于染色体整合位点分析。随后，我们将使用 ATAC-Seq和RNA-Seq用于表征基因的染色质可及性和转录活性 携带完整的前病毒（特异性目标2），确定与 完整的前病毒储库（具体目标3），并研究与完整的前病毒储库相关的表观遗传特征。 HIV-1感染阿片类药物成瘾者和HIV-1患者对照队列的储库持续性（特定目标4） 过去或现在都没有吸毒史总之，这些研究将为发展中国家提供丰富的信息。 有针对性的干预措施，以减少艾滋病毒-1感染者在抗逆转录病毒治疗期间的艾滋病毒-1持续存在， 使用阿片类药物。

Abstract Abstract Individuals who use injection drugs are among those at highest risk for HIV-1 infection, and their relative contribution to the total number of HIV-1-infected persons is increasing worldwide. This is particularly true for the roughly 2 million individuals affected by the opioid crisis in the US, of who approximately 9% (180,000 persons) are currently estimated to be HIV-1-infected. Owing to recent advances in improving access to care and adherence to treatment, a considerable proportion of these individuals is now able to maintain undetectable viral loads during ongoing use of opioids (oxycodone, heroin, hydromorphone, fentanyl) or opioid substitution agents (methadone or buprenorphine). However, residual reservoirs of virally infected cells persist in these patients, and represent the main barrier against a long-lasting drug-free remission of viral infection. Recently, there is substantial progress in understanding the cellular compartments and mechanisms of viral reservoir persistence, but opioid addicts were either highly underrepresented or entirely excluded from such investigations. Yet, there are reasons to believe that the size, structure and composition of the viral reservoir in opioid users is substantially different from HIV-1-infected individuals who do not use drugs. For instance, opioid drug abuse can profoundly change gene expression patterns and also induces epigenetic chromatin modifications, both of which are known to affect the susceptibility to retroviral infection, the selection of chromosomal integration sites and the transcriptional activity of integrated HIV-1 proviruses. This project sets out to conduct a detailed analysis of the viral reservoir structure and composition in HIV-1-infected opioid drug addicts, using a spectrum of novel next- generation sequencing technologies allowing to profile the viral reservoir at a previously unprecedented breadth and depth. In Specific Aim 1, we will comprehensively analyze the chromosomal location of intact HIV-1 proviruses, based on a novel experimental approach combining full-genome amplification, near full-length viral sequencing and ligation-mediated PCR for chromosomal integration site analysis. Subsequently, we will use ATAC-Seq and RNA-Seq to characterize the chromatin accessibility and transcriptional activity of genes harboring intact proviruses (Specific Aim 2), determine innate and adaptive immune responses correlated with the intact proviral reservoirs (Specific Aim 3), and investigate epigenetic features associated with intact proviral reservoir persistence (Specific Aim 4) in HIV-1-infected opioid addicts and a control cohort of HIV-1 patients without past or present drug abuse. Together, these studies will provide a wealth of information for developing targeted interventions to reduce HIV-1 persistence during antiretroviral therapy in HIV-1-infected individuals who use opioids.