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

骨髓吞噬细胞中 HIV DNA 中看不见的 U/A 碱基对的命运

基本信息

批准号：
9138025
负责人：
JAMES T. STIVERS
金额：
$ 40.15万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9138025
关键词：
Acquired Immunodeficiency Syndrome Adenine Affect Alveolar Alveolar Macrophages Anti-Inflammatory Agents Anti-inflammatory Base Excision Repairs Base Pairing Biochemical Bone Marrow Bronchial Lavages CD4 Positive T Lymphocytes CMV promoter Cells Characteristics Chromatin Code Cytosine DNA DNA Fragmentation DNA Repair DNA Sequence DNA-Binding Proteins Deoxyuridine Detection Drug Targeting Elements Environment Enzymes Epigenetic Process Excision Gene Expression Genetic Transcription Genetic Variation Genome Goals HIV HIV Infections HIV-1 Human Immune Immunology In Vitro Infection Inflammatory Intervention Irrigation Knowledge Life Lung Methods Mutagenesis Mutation Myelogenous Myeloid Cells Nuclear Outcome Pathway interactions Patients Phagocytes Proviruses RNA-Directed DNA Polymerase Reading Reporting Rest Reverse Transcription Role Route Sampling Scientist System T-Lymphocyte Testing Time Tissues Up-Regulation Uracil Viral Virus Virus Diseases Work base cell type combat cytokine enzyme activity knock-down macrophage monocyte novel overexpression peripheral blood promoter public health relevance repair enzyme repaired restoration small molecule time use uracil-DNA glycosylase viral DNA volunteer

项目摘要

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.