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Role of Apobecs in Retroviral Immunity

Apobecs 在逆转录病毒免疫中的作用

基本信息

批准号：
10220683
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
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-25 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220683
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 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 Lentivirus 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 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 mouse genetics multicatalytic endopeptidase complex mutant pathogen polypeptide signal peptidase tissue culture transition mutation tumor ubiquitin-protein ligase viral transmission

项目摘要

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.

项目概要先天免疫需要抑制多种病原体复制的细胞限制因子。一组限制因子是载脂蛋白 B mRNA 编辑酶，催化多肽样 (Apobec) 胞嘧啶脱氨酶家族，包括激活诱导的胞嘧啶脱氨酶 (AID)。援助由于其在控制逆转录转座子中的作用，其功能包括先天性和适应性免疫反应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 家族酶是复杂人类逆转录病毒（例如人类慢病毒）的已知抑制剂免疫缺陷病毒 (HIV-1) 以及含有 DNA 的病毒。 HIV-1 编码 Apobec 拮抗剂， Vif 以及缺乏 Vif 的情况下，原病毒由于胞嘧啶而维持大量 G 至 A 超突变逆转录过程中负链上的脱氨基作用。我的实验室表明老鼠乳腺肿瘤病毒（MMTV）是唯一已知的具有组织和功能的复杂小鼠逆转录病毒。功能特征与HIV相似，例如调节蛋白Rem。 Rem 是一种前体蛋白在内质网膜上被信号肽酶切割，产生具有 Rev 样的 N 端信号肽 (SP) 功能和未知功能的 C 末端蛋白。接种缺乏 Rem 的克隆 MMTV 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 原病毒的特征表明两条 DNA 链上广泛的 G 到 A 以及其他过渡突变，与 AID 型一致突变，以及鼠 Apobec3 的一些典型共有序列和链特异性 (mA3) 或其他脱氨酶。转染实验揭示了 AID 的蛋白酶体降解 Rem 存在，但 mA3 不存在。这些结果使我们得出这样的假设：Rem 是一种 HIV-1 Vif 样病毒多种 Apobec 胞嘧啶脱氨酶的拮抗剂。该应用程序将测试此的几个方面假设。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 或与 AID 泛素缀合来检测相互作用蛋白的生物素化的筛选融合蛋白。将研究 Rem 逆转位对 AID 降解的作用。在目标 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感染缺乏一种或多种 Apobec 基因的敲除小鼠后，在原病毒中观察到这一现象。这些研究将使用复杂的小鼠逆转录病毒来探索难以在体内测试的先天免疫的各个方面与人类病毒。我们的实验将加深对 Apobec 功能以及其在治疗人类传染病和癌症方面的潜在应用。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

APOBECs: Our fickle friends?

DOI：
10.1371/journal.ppat.1011364
发表时间：
2023-05
期刊：
PLoS pathogens
影响因子：
6.7
作者：
通讯作者：

The Role of APOBECs in Viral Replication.

DOI：
10.3390/microorganisms8121899
发表时间：
2020-11-30
期刊：
Microorganisms
影响因子：
4.5
作者：
Xu WK;Byun H;Dudley JP
通讯作者：
Dudley JP

A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus.

由复杂小鼠逆转录病毒编码的激活诱导胞苷脱氨酶的蛋白质拮抗剂。

DOI：
10.1128/mbio.01678-19
发表时间：
2019
期刊：
mBio
影响因子：
6.4
作者：
Singh,GurvaniB;Byun,Hyewon;Ali,AlmasF;Medina,Frank;Wylie,Dennis;Shivram,Haridha;Nash,AndreaK;Lozano,MaryM;Dudley,JaquelinP
通讯作者：
Dudley,JaquelinP

How Viruses Use the VCP/p97 ATPase Molecular Machine.