Fate of Invisible U/A Base Pairs Within HIV DNA in Myeloid Phagocytic Cells

 描述（由申请人提供）：我们描述了一种新型HIV-1限制/持久性机制的要素，该机制仅在骨髓来源的单核细胞（MC）和单核细胞来源的巨噬细胞（MDM）的独特细胞内环境中发挥作用，而不是静息或活化的T细胞。该机制集中在MC和MDM中的高浓度dUTP，其迫使逆转录酶（RTase）在逆转录过程中与腺嘌呤相反地掺入dUMP，导致高水平的U/A碱基对（“尿嘧啶化”）。重要的是，U/A对不会干扰编码序列，并且对于标准DNA测序方法是不可见的，因为U读作T。然而，U/A对是宿主尿嘧啶碱基切除修复（UBER）机制的底物，该机制调节这些细胞中HIV感染的结果。尿嘧啶化和UBER以不同的方式影响巨噬细胞的HIV感染。首先，通过尿嘧啶切除和病毒DNA片段化发生有效的整合前限制。逃脱初始限制的前病毒含有可以持续存在的U/A对，导致尿嘧啶诱导的HIV-1基因表达沉默。在用促炎性细胞因子刺激感染的MDM后，原本稳定的尿嘧啶化前病毒通过易错UBER变得严重诱变，表明巨噬细胞的M1/M2极化状态可以决定病毒尿嘧啶的命运。尿嘧啶化前病毒即使在数周后仍能产生低水平的感染性病毒。在三个目标中，我们寻求（i）阐明UBER途径在巨噬细胞和MC的HIV感染中的影响。(ii)确定U/A碱基对对MDM中HIV LTR驱动的转录的影响，以及（iii）使用尿嘧啶去除的时间框架及其特征性诱变特征来确定先前在接受cART的HIV感染患者的短寿命循环MC中检测到的前病毒是否来源于其与各种组织中感染的巨噬细胞的接触。阐明宿主UBER在巨噬细胞和单核细胞病毒感染中的作用，有望发现新的宿主药物靶点，用于对抗HIV在这些独特的免疫细胞中的建立和持久性。该提案汇集了临床医生和基础科学家，他们在免疫学，HIV潜伏期，尿嘧啶DNA修复和从HIV感染志愿者的支气管灌洗液中获得肺泡巨噬细胞方面具有广泛和高度互补的背景。

 DESCRIPTION (provided by applicant): We have delineated the elements of a novel HIV-1 restriction/persistence mechanism that operates solely within the unique intracellular environment of bone marrow-derived monocytes (MCs) and monocyte-derived macrophages (MDMs), but not resting or activated T cells. The mechanism centers on the high concentration of dUTP in MCs and MDMs, which forces reverse transcriptase (RTase) to incorporate dUMP opposite to adenine during reverse transcription, leading to high levels of U/A base pairs ("uracilation"). Importantly, U/A pairs do not perturb the coding sequence and are invisible to standard DNA sequencing methods because U reads as T. However, U/A pairs are a substrate for the host uracil base excision repair (UBER) machinery, which modulates the outcome of HIV infection in these cells. Uracilation and UBER impact HIV infection of macrophages in diverse ways. First, potent pre- integration restriction occurs by uracil excision and viral DNA fragmentation. Proviruses that escape initial restriction contain U/A pairs that can persist, resulting in uracil- induced silencing of HIV-1 gene expression. Upon stimulation of infected MDMs with pro-inflammatory cytokines, otherwise stable uracilated proviruses become heavily mutagenized through error-prone UBER, suggesting that the M1/M2 polarization state of the macrophage can determine the fate viral uracils. Low levels of infectious virus can emerge from uracilated proviruses even after many weeks. In three aims we seek to (i) Elucidate the impact of the UBER pathway in HIV infection of macrophages and MCs. (ii) Determine the effects of U/A base pairs on HIV LTR-driven transcription in MDMs, and (iii) Use the time frame for uracil removal and its characteristic mutagenic signature to determine whether proviruses that have been previously detected in short-lived circulating MCs in HIV infected patients on cART originate from their contact with infected macrophages in various tissues. The elucidation of the role of host UBER in viral infections of macrophages and monocytes is expected to uncover new host drug targets for combating HIV establishment and persistence in these unique immune cells. This proposal brings together clinicians and basic scientists with broad and highly complementary backgrounds in immunology, HIV latency, uracil DNA repair and in obtaining alveolar macrophages from bronchial lavage of HIV-infected volunteers.