Role of Apobecs in Retroviral Immunity

Apobecs 在逆转录病毒免疫中的作用

• 批准号: 9756136
• 负责人: Jaquelin Page Dudley
• 金额: $ 45.28万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-25 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756136
• 关键词: Affect; B-Lymphocytes; Betaretrovirus; Biological Assay; Biotinylation; C-terminal; Capsid; Cell Culture Techniques; Cells; Chimeric Proteins; Cleaved cell; Communicable Diseases; Complex; Consensus Sequence; Cullin Proteins; Cytosine; Cytosine deaminase; DNA; Data; Deaminase; Deamination; Dendritic Cells; Development; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Enzymes; Family; Family member; Genes; Genetic; Growth; HIV; HIV-1; Hepatitis B; Herpesviridae; Human; Immune response; Immunity; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; In Vitro; Inbred BALB C Mice; Incidence; Induced Mutation; Infection; Infectious Agent; Innate Immune Response; Knock-out; Knockout Mice; Laboratories; Link; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Milk; Mouse Mammary Tumor Virus; Mus; Mutation; N-terminal; Natural Immunity; Newborn Infant; Organizational Models; Papillomavirus; Peptide Signal Sequences; Proteasome Inhibitor; Protein Precursors; Proteins; Proviruses; RNA; RNA Splicing; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; Specificity; Subfamily lentivirinae; T-Lymphocyte; Technology; Testing; Transfection; Translating; Ubiquitin; Ubiquitination; Uracil; Variant; Viral; Virion; Virus; Virus Replication; Wild Type Mouse; activation-induced cytidine deaminase; adaptive immune response; adaptive immunity; apolipoprotein B mRNA editing enzyme; base; cell type; env Gene Products; experimental study; genetic regulatory protein; human disease; improved; in vivo; in vivo evaluation; inhibitor/antagonist; malignant breast neoplasm; member; multicatalytic endopeptidase complex; mutant; pathogen; polypeptide; signal peptidase; tissue culture; transition mutation; tumor; ubiquitin-protein ligase; viral transmission

Project Summary Innate immunity requires cellular restriction factors that inhibit the replication of multiple pathogens. One group of restriction factors is the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (Apobec) family of cytosine deaminases, including activation-induced cytosine deaminase (AID). AID function includes both innate and adaptive immune responses due to its role in control of retrotransposons as well as somatic hypermutation of immunoglobulin variable regions and class switch recombination. Apobec family enzymes are known inhibitors of complex human retroviruses, such as the lentivirus human immunodeficiency virus (HIV-1), as well as DNA-containing viruses. HIV-1 encodes an Apobec antagonist, Vif and, in the absence of Vif, the provirus sustains high numbers of G to A hypermutations due to cytosine deamination on the minus strand during reverse transcription. My laboratory has shown that mouse mammary tumor virus (MMTV) is the only known complex mouse retrovirus with organizational and functional features similar to HIV, such as the regulatory protein Rem. Rem is a precursor protein that is cleaved by signal peptidase at the ER membrane to give an N-terminal signal peptide (SP) that has Rev-like function and a C-terminal protein of unknown function. Inoculation of cloned MMTV lacking Rem expression into BALB/c mice resulted in mammary tumors with a decreased incidence and latency compared to wild-type MMTV. Characterization of MMTV proviruses from these mammary tumors showed extensive G to A as well as other transition mutations on both DNA strands, consistent with AID-type mutations, but also some typical of the consensus sequences and strand specificity of murine Apobec3 (mA3) or other deaminases. Transfection experiments revealed proteasomal degradation of AID in the presence of Rem, but not mA3. These results led us to the hypothesis that Rem is an HIV-1 Vif-like antagonist of multiple Apobec cytosine deaminases. This application will test several aspects of this hypothesis. In Aim 1, we will determine sequences within Rem and AID that are necessary for proteasomal degradation. We will identify the E3 ligase for AID protein interactions in the presence and absence of Rem using screens that detect biotinylation of interacting proteins by BirA or conjugation to an AID-ubiquitin fusion protein. The role of Rem retrotranslocation for AID degradation will be investigated. In Aim 2, MMTVs or T-cell variants lacking Rem expression will be used to determine the types of transition mutations observed in proviruses after infections of knockout mice lacking one or more Apobec genes. These studies will use a complex mouse retrovirus to explore aspects of innate immunity that are difficult to test in vivo with human viruses. Our experiments will provide improved understanding of Apobec function as well as potential application to the treatment of human infectious diseases and cancers.

项目摘要 先天免疫需要细胞限制因子来抑制多种病原体的复制。 一组限制性因子是载脂蛋白B mRNA编辑酶，催化多肽样酶， （Apobec）胞嘧啶脱氨酶家族，包括活化诱导的胞嘧啶脱氨酶（AID）。援助 由于其在逆转录转座子控制中的作用，其功能包括先天性和适应性免疫应答 以及免疫球蛋白可变区的体细胞超突变和类别转换重组。 Apobec家族酶是复杂的人逆转录病毒（如慢病毒人逆转录病毒）的已知抑制剂。 免疫缺陷病毒（HIV-1）以及含DNA的病毒。HIV-1编码Apobec拮抗剂， Vif，并且在Vif缺失的情况下，由于胞嘧啶，前病毒维持高数量的G到A超突变。 反转录过程中负链的脱氨作用。我的实验室已经证明老鼠 乳腺肿瘤病毒（MMTV）是唯一已知的复杂的小鼠逆转录病毒， 功能特征类似于HIV，如调节蛋白Rem。Rem是一种前体蛋白， 在ER膜上被信号肽酶切割，得到N-末端信号肽（SP）， 功能和未知功能的C-末端蛋白质。接种缺乏Rem的克隆MMTV 在BALB/c小鼠中的表达导致乳腺肿瘤的发生率和潜伏期降低 与野生型MMTV相比。来自这些乳腺肿瘤的MMTV前病毒的特征显示 广泛的G到A以及两条DNA链上的其他转换突变，与艾滋病类型一致 突变，但也有一些典型的共有序列和链特异性的小鼠Apobec 3 (mA3)或其它脱氨酶。转染实验表明，在细胞中AID的蛋白酶体降解。 存在Rem，但不存在mA 3。这些结果使我们假设Rem是一种HIV-1 Vif样蛋白， 多种Apobec胞嘧啶脱氨酶的拮抗剂。这个应用程序将测试几个方面 假说.在目标1中，我们将确定Rem和AID中蛋白酶体必需的序列， 降解我们将鉴定在存在和不存在Rem的情况下AID蛋白相互作用的E3连接酶 使用通过BirA检测相互作用蛋白质的生物素化或与AID-泛素缀合的筛选 融合蛋白将研究Rem逆易位对AID降解的作用。在目标2中， MMTV或缺乏Rem表达的T细胞变体将用于确定转换突变的类型 在缺乏一个或多个Apobec基因的敲除小鼠感染后的前病毒中观察到。这些研究 将使用一种复杂的小鼠逆转录病毒来探索先天免疫的各个方面，这些方面很难在体内进行测试 人类病毒。我们的实验将提供对Apobec功能的更好理解， 在治疗人类感染性疾病和癌症中的潜在应用。