Retroviral Subversion of ERAD and Intrinsic Immunity

描述(申请人提供)：阿尔茨海默氏症、囊性纤维化、传染病和癌症都有一个共同的特征：不能有效地破坏错误折叠或组装的蛋白质。跨膜和分泌蛋白使用信号肽(SP)直接翻译到内质网(ER)，在内质网中容易发生错误的蛋白质折叠和修饰过程。ER相关降解(ERAD)过程识别错误折叠的蛋白质，导致使用E1、E2和E3酶添加泛素链。然后，从内质网膜上提取泛素化的多聚蛋白，这一过程被称为逆转位或错位，并被输送到胞浆蛋白酶体进行降解。包括病毒和细菌在内的许多病原体利用ERAD进一步传播。小鼠乳腺肿瘤病毒(MMTV)是一种利用ERAD复制和诱导乳腺癌和白血病的复杂逆转录病毒。MMTV产生一种调节蛋白(Rem)，它由一个包含HIV Rev.所有功能基序的长信号肽(Rem-SP)导向内质网。Rem被内质网管腔中的信号肽酶切割，产生N-末端Rem-SP和一个独特的C-末端(RemCT)。在一条史无前例的途径中，Rem-SP被反向移位到胞浆，在那里它避免了在核输入之前蛋白酶体的降解。然后，REM-SP与病毒RNA结合，以实现高效的核输出和表达。尽管逆转位被认为需要靶蛋白的多泛素化，但我们的初步数据表明，用于所有已知ERAD底物的E酶对于Rem-SP逆转录易位是必不可少的。我们的假设是，Rem-SP使用一种以前未知的过程，不需要多泛素化或E1酶Uba1进行逆转录易位。在第一个具体目标中，将使用两种方法来识别涉及ERAD颠覆的细胞组件。Rem的C-末端GFP标记阻止了逆转位，并将用于纯化逆转位中间体和相关的细胞蛋白，以便进行质谱分析。另一种方法将使用基于荧光的报告分析和小发夹文库来识别参与ERAD和Rem运输的新细胞蛋白。在第二个特定目标中，已经提出了令人兴奋的初步数据，表明与野生型前病毒相比，缺乏RemCT表达的MMTV前病毒具有更多的G到A突变。Rem和激活诱导型胞苷脱氨酶(AID)的共表达可导致AID的降解。AID是一种已知的抗体体细胞超突变和类开关重组所必需的诱变剂。我们认为MMTV是第一个对抗AID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RemCT的定位及其在细胞培养和体内基因敲除小鼠中对抗细胞胞苷脱氨酶的能力来检验。进一步了解ERAD和固有免疫对于开发癌症和病原性病毒感染的新治疗方法至关重要。 公共卫生相关性：内质网相关降解(ERAD)是破坏传染病、阿尔茨海默病、囊性纤维化和癌症中发现的许多有缺陷的细胞蛋白所必需的，但可被许多病原体破坏，包括致癌逆转录病毒小鼠乳腺肿瘤病毒(MMTV)。MMTV编码的Rem蛋白使用ERAD来控制病毒蛋白的表达，似乎是第一个拮抗激活诱导的胞苷脱氨酶(AID)蛋白的病毒蛋白，AID蛋白会导致双链DNA断裂和人类癌症。对ERAD和固有免疫的研究以及病原体对它们的开发对于开发许多人类疾病的新的有效治疗方法是必不可少的。

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.