Persistence and Fate of Invisible U/A Pairs in HIV-1 Proviral DNA

描述(由申请人提供)：对HIV的内在免疫反应中一个知之甚少的分子是简单核苷酸脱氧尿苷三磷酸(DUTP)。在非复制型免疫细胞中，dUTP水平通常很高，在感染HIV后，病毒逆转录酶将dUTP整合到新生病毒基因组中，产生U/A碱基对，而不是正常的Watson-Crick T/A对。在缺乏核尿嘧啶碱基切除修复(Uber)的情况下，大量尿酸碱基切除修复的病毒DNA完全有能力整合，但会沉默病毒基因的表达。由于Uber在静息细胞中几乎检测不到，这些发现表明尿嘧啶前驱物将持续存在，直到静息细胞激活。这些考虑导致我们提出，持续的U/A碱基对在HIV-1潜伏期和重新激活中扮演着以前未被认识到的角色。为了表征HIV感染的这一方面，我们将使用一种新的下一代DNA测序方法(BE-SEQ)来绘制Uber缺陷细胞系中未整合和整合的HIV-1 DNA中“看不见的”U/A碱基对的空间分布和时间命运。高分辨率的尿嘧啶作图将提供有价值的机制信息，帮助我们了解dUTP如何通过逆转录酶(RTase)整合到负链和正链DNA产物中，以及它的空间分布、持久性和可能容易出错的移除可能如何影响Uber缺陷免疫细胞中的病毒持久性。然后，我们将确定从HIV-1感染患者的静息CD4+T细胞中获得的前病毒DNA中，dUTP掺入或酶促胞嘧啶脱氨所产生的尿嘧啶的程度和持久性。然后，这些静止的T细胞将在体外被激活，并将使用一种新的跨井病毒生长试验来评估前病毒尿嘧啶的命运。在静息状态下沉默病毒基因转录的隐形尿酸可以在重新激活后修复，或者相反，导致致命的 容易出错的修复机制导致的基因改变。总体而言，这些研究将确定前病毒DNA序列中U/A对的分布、持久性和命运，以及它们对病毒潜伏期和感染的潜在影响。

DESCRIPTION (provided by applicant): A poorly understood molecular player in the intrinsic immune response against HIV is the simple nucleotide deoxyuridine triphosphate (dUTP). In non-replicating immune cells, dUTP levels are typically high, and upon HIV infection, viral reverse transcriptase incorporates dUTP into the nascent viral genome to generate U/A base pairs instead of the normal Watson-Crick T/A pairs. In the absence of nuclear uracil base excision repair (UBER) heavily uracilated viral DNA is fully competent for integration, but silences viral gene expression. Because UBER is virtually undetectable in resting cells, these findings indicate that uracilated proviruses will persist until a resting cell is activated. These considerations lead us to propose that persistent U/A base pairs play a previously unrecognized role in HIV-1 latency and reactivation. To characterize this aspect of HIV infection we will use a novel next-generation DNA sequencing method (BE-SEQ) to map the spatial distribution and temporal fate of "invisible" U/A base pairs in unintegrated and integrated HIV-1 DNA in a UBER-deficient cell line. High-resolution uracil mapping will provide valuable mechanistic information t understand how dUTP is incorporated into minus and plus strand DNA products by reverse transcriptase (RTase), and how its spatial distribution, persistence and possible error-prone removal may impact viral persistence in UBER-deficient immune cells. We will then determine the extent and persistence of uracil generated from dUTP incorporation or enzymatic cytosine deamination in proviral DNA obtained from resting CD4+ T cells of HIV-1 infected patients. These resting T cells will then be activated in vitro, and the fate of proviral uracils will be evaluated using a new trans-well viral outgrowth assay. Invisible uracils that silence viral gene transcription in the resting state could be repaired after reactivation, or instead, lead to lethal genetic alterations by error prone repair mechanisms. Overall, these studies will determine the distribution, persistence and fate of U/A pairs in proviral DNA sequences and their potential effect on viral latency and infection.