Zhang J, Chen H, Weinmaster G, Hayward S D
Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, USA.
J Virol. 2001 Mar;75(6):2946-56. doi: 10.1128/JVI.75.6.2946-2956.2001.
The Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione S-transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional interaction between RPMS and corepressor proteins. GST affinity assays revealed interaction between RPMS and the corepressor Sin3A and CIR. The RPMS-CIR interaction was further substantiated in mammalian two-hybrid, coimmunoprecipitation, and colocalization experiments. RPMS has been shown to interfere with NotchIC and EBNA2 activation of CBF1-containing promoters in reporter assays. Consistent with this function, immunofluorescence assays performed on cotransfected cells showed that there was colocalization of RPMS with NotchIC and with EBNA2 in intranuclear punctate speckles. The effect of RPMS on NotchIC function was further examined in a muscle cell differentiation assay where RPMS was found to partially reverse NotchIC-mediated inhibition of differentiation. The mechanism of RPMS action was examined in cotransfection and mammalian two-hybrid assays. The results revealed that RPMS blocked relief of CBF1-mediated repression and interfered with SKIP-CIR interactions. We conclude that RPMS acts as a negative regulator of EBNA2 and Notch activity through its interactions with the CBF1-associated corepressor complex.
爱泼斯坦-巴尔病毒（EBV）的BamHI-A向右转录本（BARTs）在所有与EBV相关的肿瘤中均有表达，在体内潜伏感染的B细胞以及培养的B细胞系中也有表达。BART家族转录本之一包含一个开放阅读框RPMS1，其编码一种名为RPMS的核蛋白。逆转录-聚合酶链反应分析显示，具有产生RPMS1开放阅读框剪接模式的BART转录本在EBV阳性淋巴母细胞系中普遍表达，在霍奇金病组织中也能检测到。为确定RPMS功能而进行的实验表明，RPMS与CBF1以及CBF1相关共抑制复合物的成分相互作用。在谷胱甘肽S-转移酶（GST）亲和试验中证实了RPMS与CBF1的相互作用，并且RPMS能够改变突变型CBF1的细胞内定位。Gal4-RPMS融合蛋白介导转录抑制，提示RPMS与共抑制蛋白之间存在额外的相互作用。GST亲和试验显示RPMS与共抑制因子Sin3A和CIR之间存在相互作用。在哺乳动物双杂交、免疫共沉淀和共定位实验中进一步证实了RPMS与CIR的相互作用。在报告基因试验中，已证明RPMS会干扰NotchIC和EBNA2对含CBF1启动子的激活。与此功能一致，对共转染细胞进行的免疫荧光试验表明，RPMS与NotchIC以及EBNA2在核内点状斑点中共定位。在肌肉细胞分化试验中进一步研究了RPMS对NotchIC功能的影响，发现RPMS能部分逆转NotchIC介导的分化抑制。在共转染和哺乳动物双杂交试验中研究了RPMS的作用机制。结果显示，RPMS阻断了CBF1介导的抑制作用的解除，并干扰了SKIP与CIR的相互作用。我们得出结论，RPMS通过与CBF1相关共抑制复合物的相互作用，作为EBNA2和Notch活性的负调节因子发挥作用。
