Retroviral Subversion of ERAD and Intrinsic Immunity

ERAD 和内在免疫的逆转录病毒颠覆

• 批准号: 8438721
• 负责人: Jaquelin Page Dudley
• 金额: $ 37.09万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-10 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8438721
• 关键词: Affect; Alzheimer' s Disease; Amino Acids; Antibodies; Antibody Formation; Area; B-Cell Neoplasm; B-Lymphocytes; Bacteria; Binding; Biological Assay; C-terminal; Cations; Cell Culture Techniques; Cell Nucleus; Cells; Chromosomal translocation; Cleaved cell; Communicable Diseases; Complex; Cystic Fibrosis; Cytidine Deaminase; Cytidine Deaminase Inhibitor; Cytosol; DNA Double Strand Break; Data; Development; Disease; Dislocations; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Engineering; Enzymes; Failure; Family; Family member; Fluorescence; Genetic Transcription; Genome; Goals; HIV; Hormones; Human; Immune response; Immunity; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Infection; Knockout Mice; Knowledge; Length; Libraries; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Modeling; Molecular Chaperones; Monitor; Mouse Mammary Tumor Virus; Mus; Mutagens; Mutation; N-terminal; Nonsense Codon; Nuclear Export; Nuclear Import; Oncogenic; Oncogenic Viruses; Paper; Pathway interactions; Peptide Signal Sequences; Peptides; Phenotype; Plasmids; Polyubiquitination; Post-Translational Protein Processing; Process; Production; Protein Analysis; Proteins; Proviruses; RNA; Regulatory Pathway; Reporter; Retroviridae; Scheme; Series; Signal Transduction; Superantigens; Terminator Codon; Testing; Translations; Ubiquitin; Ubiquitin-Activating Enzymes; Viral; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; activation-induced cytidine deaminase; base; cancer therapy; effective therapy; genetic regulatory protein; human disease; in vivo; innovation; leukemia; malignant breast neoplasm; multicatalytic endopeptidase complex; mutant; novel; pathogen; prevent; protein expression; protein folding; protein misfolding; signal peptidase; trafficking; transmission process; viral RNA

DESCRIPTION (provided by applicant): Alzheimer's, cystic fibrosis, infectious diseases, and cancer all have a common feature: failure to efficiently destroy incorrectly folded or assembled proteins. Transmembrane and secreted proteins use a signal peptide (SP) to direct translation to the endoplasmic reticulum (ER), where an error-prone process of protein folding and modification occur. The ER-associated degradation (ERAD) process identifies misfolded proteins, leading to addition of ubiquitin chains using E1, E2, and E3 enzymes. Polyubiquitinated proteins then are extracted from the ER membrane, a process known as retrotranslocation or dislocation, and delivered to cytosolic proteasomes for degradation. Many pathogens, including viruses and bacteria, exploit ERAD to further their dissemination. Mouse mammary tumor virus (MMTV) is a complex retrovirus that uses ERAD for replication and induction of breast cancer and leukemia. MMTV produces a regulatory protein (Rem), which is directed to the ER by a long signal peptide (Rem-SP) containing all the functional motifs found in HIV Rev. Rem is cleaved by signal peptidase in the ER lumen to yield N-terminal Rem-SP and a unique C-terminus (RemCT). In an unprecedented pathway, Rem-SP is retrotranslocated to the cytosol, where it avoids proteasomal degradation prior to nuclear import. Rem-SP then binds viral RNA to allow efficient nuclear export and expression. Although retrotranslocation is believed to require polyubiquitination of target proteins, our preliminary data indicate that the E enzyme used for all known ERAD substrates is dispensable for Rem-SP retrotranslocation. Our hypothesis is that Rem-SP uses a previously unknown process that does not require polyubiquitination or the E1 enzyme Uba1 for retrotranslocation. In the first specific aim, two approaches will be used to identify cellular components involved in subversion of ERAD. C-terminal GFP tagging of Rem blocks retrotranslocation and will be used to purify a retrotranslocation intermediate and associated cellular proteins for analysis by mass spectrometry. An alternative approach will use a fluorescence-based reporter assay and a small-hairpin library to identify new cellular proteins involved in ERAD and Rem trafficking. In the second specific aim, exciting preliminary data have been presented showing that MMTV proviruses lacking RemCT expression have many G to A mutations compared to wild-type proviruses. Co-expression of Rem and activation-induced cytidine deaminase (AID), a known mutagen required for antibody somatic hypermutation and class switch recombination, results in AID degradation. We propose that MMTV is the first virus to antagonize AID to avoid both viral genome mutation and maturation of the MMTV-specific antibody response. This idea will be tested by monitoring RemCT localization and its ability to antagonize cellular cytidine deaminases both in cell culture and in knockout mice in vivo. Further understanding of ERAD and intrinsic immunity are essential to develop new treatments for cancer and pathogenic viral infections. PUBLIC HEALTH RELEVANCE: Endoplasmic reticulum-associated degradation (ERAD) is essential for destruction of many defective cellular proteins found in infectious diseases, Alzheimer's disease, cystic fibrosis, and cancer, but is subverted by a number of pathogens, including the oncogenic retrovirus mouse mammary tumor virus (MMTV). The MMTV-encoded Rem protein uses ERAD to control viral protein expression and appears to be the first viral protein that antagonizes the activation-induced cytidine deaminase (AID) protein, which causes double- stranded DNA breaks and human cancers. Studies of ERAD and intrinsic immunity and their exploitation by pathogens are essential for development of new and effective treatments for many human diseases.

描述（由申请人提供）：阿尔茨海默氏症，囊性纤维化，传染病和癌症都具有共同特征：未能有效破坏错误折叠或组装的蛋白质。跨膜和分泌的蛋白使用信号肽（SP）将转化为内质网（ER），其中发生了容易发生的蛋白质折叠和修饰过程。与ER相关的降解（ERAD）过程鉴定了错误折叠的蛋白质，从而导致使用E1，E2和E3酶添加泛素链。然后从ER膜中提取多泛素化的蛋白质，该过程称为逆转录或脱位，并递送至胞质蛋白酶体降解。许多病原体，包括病毒和细菌，都利用Erad进一步传播。小鼠乳腺肿瘤病毒（MMTV）是一种复杂的逆转录病毒，使用ERAD复制和诱导乳腺癌和白血病。 MMTV产生一种调节蛋白（REM），该蛋白是由长的信号肽（REM-SP）指向ER的，其中包含HIV Rev中发现的所有功能基序。REM被ER中的信号肽酶在ER腔中裂解以产生N端REM-SP和独特的C-末端（REMCT）。在空前的途径中，将REM-SP递归转换为细胞质，在核进口之前避免了蛋白酶体降解。然后，REM SP结合病毒RNA，以允许有效的核输出和表达。尽管据信逆转录趋化性需要对靶蛋白进行多泛素化，但我们的初步数据表明，用于所有已知ERAD底物的E酶是可用于REM-SP逆转录液的可分配。我们的假设是，REM-SP使用以前未知的过程，该过程不需要多泛素化或E1酶UBA1进行逆转录。在第一个特定目标中，将使用两种方法来识别与Erad颠覆有关的细胞成分。 REM块的C末端GFP标记逆转录分配，并将用于净化反转换中间体和相关的细胞蛋白，以通过质谱法分析。另一种方法将使用基于荧光的记者测定法和小发质库来鉴定与ERAD和REM运输有关的新细胞蛋白。在第二个特定目标中，已经提出了令人兴奋的初步数据，表明与野生型预科病毒相比，缺乏REMCT表达的MMTV前期病毒与突变具有许多G。 REM和激活诱导的胞苷脱氨酶（AID）的共表达，这是一种已知的抗体体细胞超偏变和类转换重组所需的诱变，导致辅助降解。我们建议MMTV是第一个与辅助拮抗的病毒，以避免病毒基因组突变和MMTV特异性抗体反应的成熟。这一想法将通过监测转置定位及其在体内拮抗细胞培养和基因敲除小鼠中拮抗细胞胞苷脱氨酶的能力来测试。对ERAD和内在免疫力的进一步了解对于开发新的治疗癌症和致病病毒感染至关重要。 PUBLIC HEALTH RELEVANCE: Endoplasmic reticulum-associated degradation (ERAD) is essential for destruction of many defective cellular proteins found in infectious diseases, Alzheimer's disease, cystic fibrosis, and cancer, but is subverted by a number of pathogens, including the oncogenic retrovirus mouse mammary tumor virus (MMTV). MMTV编码的REM蛋白使用ERAD来控制病毒蛋白的表达，并且似乎是拮抗激活诱导的胞苷脱氨酶（AID）蛋白的第一个病毒蛋白，该蛋白会引起双链DNA断裂和人类癌。对ERAD和内在免疫力的研究及其对病原体的剥削对于为许多人类疾病开发新的有效疗法至关重要。