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

HIV-1 前病毒 DNA 中隐形 U/A 对的持久性和命运

基本信息

批准号：
8790165
负责人：
JAMES T. STIVERS
金额：
$ 16.65万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-11 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8790165
关键词：
Acquired Immunodeficiency Syndrome Address Base Excision Repairs Base Pairing Biological Assay Biology Biomedical Research CD4 Positive T Lymphocytes Cell Line Cells Communities Coupling Cytosine DNA DNA Integration DNA Sequence Deamination Enzymes Excision Gene Expression Gene Silencing Genetic Transcription Genome Genomics HIV HIV Infections HIV-1 Highly Active Antiretroviral Therapy Human Immune Immune response In Vitro Infection Interphase Cell Intervention Lead Maps Methodology Methods Molecular Mutagenesis Mutation Nuclear Nucleotides Patients Play Population Proviruses RNA-Directed DNA Polymerase Reading Resolution Rest Role Route Spatial Distribution T-Lymphocyte Time Uracil Viral Viral Genes Viral Genome Viral Load result Virus Virus Diseases Virus Latency Work antiretroviral therapy base deoxyuridine triphosphate digital genome-wide macrophage next generation novel public health relevance reactivation from latency repaired uracil-DNA glycosylase viral DNA

项目摘要

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.

描述（由申请人提供）：在针对 HIV 的内在免疫反应中，人们知之甚少的分子参与者是简单核苷酸脱氧尿苷三磷酸 (dUTP)。在非复制免疫细胞中，dUTP 水平通常很高，并且在 HIV 感染后，病毒逆转录酶将 dUTP 整合到新生病毒基因组中，生成 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，重度尿嘧啶的病毒DNA完全能够整合，但会沉默病毒基因表达。由于 UBER 在静息细胞中几乎检测不到，因此这些发现表明尿酸原病毒将持续存在，直到静息细胞被激活。这些考虑使我们提出持久的 U/A 碱基对在 HIV-1 潜伏和重新激活中发挥着以前未被认识到的作用。为了表征 HIV 感染的这一方面，我们将使用一种新型的下一代 DNA 测序方法 (BE-SEQ) 来绘制未整合和整合的 HIV-1 DNA 中“看不见的”U/A 碱基对的空间分布和时间命运。 UBER 缺陷细胞系。高分辨率尿嘧啶图谱将提供有价值的机制信息，以了解 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去除如何影响 UBER-中的病毒持久性。免疫细胞缺陷。然后，我们将确定从 HIV-1 感染患者的静息 CD4+ T 细胞获得的前病毒 DNA 中 dUTP 掺入或酶促胞嘧啶脱氨基作用产生的尿嘧啶的程度和持久性。然后，这些静息 T 细胞将在体外被激活，并且将使用新的跨孔病毒生长测定来评估原病毒尿嘧啶的命运。在静息状态下沉默病毒基因转录的看不见的尿嘧啶可以在重新激活后被修复，或者相反，导致致命容易出错的修复机制导致的遗传改变。总体而言，这些研究将确定原病毒 DNA 序列中 U/A 对的分布、持久性和命运及其对病毒潜伏期和感染的潜在影响。