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; 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 先天免疫需要抑制多种病原体复制的细胞限制因素。 一组限制因素是载脂蛋白B mRNA编辑酶，催化多肽样酶 （APOBEC）胞嘧啶脱氨酶家族，包括激活诱导的胞嘧啶脱氨酶（AID）。援助 功能包括先天性和适应性免疫反应，因为其在控制逆转录子中的作用 以及免疫球蛋白可变区域和类开关重组的体细胞超昂贵。 APOBEC家族酶是复杂人逆转录病毒的已知抑制剂，例如慢病毒 免疫缺陷病毒（HIV-1）以及含DNA的病毒。 HIV-1编码APOBEC拮抗剂， VIF，在没有VIF的情况下，由于胞嘧啶导致的高压剂量持续数量高。 逆转录过程中负链的脱氨酸。我的实验室表明鼠标 乳腺肿瘤病毒（MMTV）是唯一具有组织和组织的复杂小鼠逆转录病毒 与HIV相似的功能特征，例如调节蛋白REM。 REM是一种前体蛋白 在ER膜上被信号肽酶裂解，得出具有转速样的N末端信号肽（SP） 功能和未知功能的C末端蛋白。接种克隆的MMTV缺乏REM 在BALB/C小鼠中的表达导致乳腺肿瘤的发病率降低和潜伏期 与野生型MMTV相比。这些乳腺肿瘤的MMTV病毒的表征显示 在两个DNA链上的广泛g以及其他过渡突变，与辅助类型一致 突变，但也是鼠APOBEC3共识序列和链特异性的典型典型 （MA3）或其他脱氨酶。转染实验表明，在 REM的存在，但不存在MA3。这些结果使我们提出了REM是HIV-1 VIF样的假设 多个Apobec胞嘧啶脱氨酶的拮抗剂。该应用程序将测试其中的几个方面 假设。在AIM 1中，我们将确定REM内的序列以及蛋白酶体所必需的辅助 降解。我们将在存在和不存在REM的情况下确定E3连接酶的AID蛋白相互作用 使用BIRA检测相互作用蛋白的生物素化或与辅助泛素结合的筛选 融合蛋白。将研究REM转递转换剂在辅助降解中的作用。在AIM 2中， 缺乏REM表达的MMTV或T细胞变体将用于确定过渡突变的类型 在缺乏一个或多个APOBEC基因的基因敲除小鼠感染后，在病毒中观察到。这些研究 将使用复杂的小鼠逆转录病毒来探索很难在体内测试的先天免疫的方面 有人类病毒。我们的实验将提高对APOBEC功能以及 潜在的应用人类传染病和癌症治疗。