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

 描述(申请人提供)：我们描述了一种新的HIV-1限制/持续机制的要素，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工作，而不是静止或激活的T细胞。其机制集中在MC和MDM中高浓度的dUTP，这迫使逆转录酶(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(“尿化”)。重要的是，U/A对不会扰乱编码序列，并且对标准的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。优步和优步以不同的方式影响巨噬细胞的艾滋病毒感染。首先，有效的整合前限制是通过切除尿嘧啶和病毒DNA碎片来实现的。逃脱最初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。在感染的MDM被促炎细胞因子刺激后，其他稳定的尿嘧啶前病毒会通过容易出错的Uber发生严重突变，这表明巨噬细胞的M1/M2极化状态可以决定病毒尿嘧啶的命运。即使在许多周后，尿毒症前病毒也可能出现低水平的传染性病毒。在三个目标中，我们试图(I)阐明Uber途径在HIV感染巨噬细胞和MC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)利用尿嘧啶清除的时间框架及其特有的突变特征来确定先前在CART上的HIV感染患者的短暂循环MC中检测到的前病毒是否起源于他们与各种组织中受感染的巨噬细胞的接触。宿主Uber在巨噬细胞和单核细胞病毒感染中的作用的阐明，有望发现新的宿主药物靶点，以对抗艾滋病毒在这些独特的免疫细胞中的建立和持续。这项建议将在免疫学、艾滋病毒潜伏期、尿嘧啶DNA修复以及从艾滋病毒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背景的临床医生和基础科学家聚集在一起。

 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.