Viral RNPs, mRNA Stability and Export

病毒 RNP、mRNA 稳定性和输出

• 批准号: 6989639
• 负责人: JOAN A. STEITZ
• 金额: $ 11.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-02 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6989639
• 关键词: B lymphocyte; Epstein Barr virus; Herpesvirus saimiri; RNA interference; T lymphocyte; Xenopus; cell transformation; crosslink; gene deletion mutation; gene expression; host organism interaction; human herpesvirus 8; messenger RNA; microarray technology; neoplasm /cancer genetics; oncogenic virus; phenotype; protein protein interaction; regulatory gene; viral carcinogenesis; virus RNA; virus protein

The roles of non-coding nuclear RNAs in lymphoid cells harboring three transforming herpesviruses are being investigated. Epstein-Barr virus (EBV) infects and transforms human B cells and is the causative agent of infectious mononucleosis, as well as being associated with several human cancers. Herpesvirus saimiri induces fatal lymphomas and leukemias in New World monkeys and transforms human and monkey T lymphocytes in culture, resulting in a mature, activated phenotype. Kaposi sarcoma-associated herpesvirus (KSHV) afflicts AIDS and other immunocompromised patients and, like other herpesviruses, persists in a latent form until activation of the lytic phase. Among the few viral gene products expressed in transformed cells are the two EBV-encoded EBERs and the seven H. saimiri-encoded HSURS; yet, they are not essential for viral growth or transformation. Upon induction, KSHV produces PAN, a capped, polyadenylated RNA that does not leave the nucleus. These viral RNAs are all relatively small (<1.1 kb), abundant, conserved, and associate with host proteins that - in the case of EBERs and HSURs - are autoantigens. They are therefore excellent probes for dissecting host cell pathways, as well as mechanisms of viral transformation or lytic growth. Proposed experiments will test both the hypothesis that EBERs, HSURs and PAN may perturb cellular metabolism by sequestering important host proteins, and the possibility that their RNPs perform some function critical for the virus. The molecular details of the interactions of protein L22, PKR and La with EBER1 will be probed by structural studies and nucleotide analog interference mapping. Components of the EBER2 RNP will be identified. EBERs will be examined for possible shuttling between the nucleus and cytoplasm, and their contribution to host gene expression assessed by microarray analyses, Microarray evidence that HSURs 1 and 2 enhance the activated state of transformed T cells will be confirmed in rescue experiments using lentiviral vectors, their interaction with TTP and its effects examined, and the level at which HSURs modulate host gene expression determined. RNA elements and trans-acting factors essential for PAN accumulation in the nucleus will be characterized. PAN function in KSHV lytic infection will be investigated by siRNA knockdown and/or genomic PAN deletion, followed by complementation experiments.

正在研究非编码核 RNA 在携带三种转化疱疹病毒的淋巴细胞中的作用。 EB 病毒 (EBV) 感染并转化人类 B 细胞，是传染性单核细胞增多症的病原体，并且与多种人类癌症有关。赛米尔疱疹病毒可在新世界猴中诱导致命性淋巴瘤和白血病，并在培养物中转化人类和猴 T 淋巴细胞，从而产生成熟的激活表型。卡波西肉瘤相关疱疹病毒 (KSHV) 折磨着艾滋病和其他免疫功能低下的患者，并且与其他疱疹病毒一样，以潜伏形式持续存在，直到激活裂解期。在转化细胞中表达的少数病毒基因产物包括两个 EBV 编码的 EBER 和七个 H. saimiri 编码的 HSURS；然而，他们是 对于病毒式增长或转化不是必需的。诱导后，KSHV 产生 PAN，一种封端的、 不离开细胞核的聚腺苷酸化RNA。这些病毒 RNA 都相对较小（<1.1 kb）、丰富、保守，并且与宿主蛋白相关（就 EBER 和 HSUR 而言），这些蛋白是自身抗原。因此，它们是剖析宿主细胞途径以及病毒转化或裂解生长机制的优秀探针。拟议的实验将测试以下假设：EBER、HSUR 和 PAN 可能通过隔离重要的宿主蛋白来扰乱细胞代谢，以及它们的 RNP 执行某些对病毒至关重要的功能的可能性。蛋白质 L22、PKR 和 La 相互作用的分子细节 EBER1将通过结构研究和核苷酸类似物干扰图谱进行探测。 EBER2 RNP 的组件将被识别。将检查 EBER 是否可能在细胞核和细胞质之间穿梭，并通过微阵列分析评估它们对宿主基因表达的贡献，微阵列证据表明 HSUR 1 和 2 增强转化 T 细胞的激活状态将在使用慢病毒载体的救援实验中得到证实，研究人员检查了它们与 TTP 的相互作用及其影响，并确定了 HSUR 调节宿主基因表达的水平。将对 PAN 在细胞核中积累所必需的 RNA 元件和反式作用因子进行表征。将通过 siRNA 敲低和/或基因组 PAN 缺失来研究 KSHV 裂解性感染中的 PAN 功能，然后进行互补实验。