The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) causes Kaposi''s sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1''s effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.     

EB病毒即刻早期BRLF1基因在淋巴瘤组织中多态性研究

目的：EB病毒（Epstein．Barrvirus,EBV）感染宿主细胞包括潜伏期和裂解期,它的即刻早期基因BRLFl编码产物Rta蛋白是EBV从潜伏期到裂解期的关键性调节因子。目前,Rta蛋白已成为治疗EBV相关肿瘤的靶蛋白,但有关BRLFl基因在EBV阳性淋巴瘤中的研究甚少,本研究旨在明确EB病毒即刻早期基因BRLFl在EB病毒阳性淋巴瘤组织中的变异规律,探讨其变异意义。方法：原位杂交筛选EB病毒阳性淋巴瘤（BL）,提取EB病毒DNA。PCR结合DNA测序检测EBV阳性淋巴瘤中BRLFl基因多态性,应用DNAStar软件对序列进行对比分析。结果：共筛选出27例EBV阳性淋巴瘤标本,在27例BL阳性标本中成功扩增BRLFl基因,与B95—8标准序列比对分析,共发现20处无义突变和19处有义突变。8例属于BR1-A亚型,18例属于BRl—C亚型,1例属于BRl-E亚型,其中以BRl-A亚型和BRl-C亚型最为常见,分别为29．6％（8／27）、66．7％（18／27）。与EBVaGC、NPC和1W相比,4种人群BRLFl亚型的分布不同,其中BRl-A亚型更多出现在健康人群中,BRl-C亚型更多出现在NPC和BL中。在Rta蛋白功能区中,二聚体区域呈高度保守,在DNA结合区域和反式激活区域均检测出序列突变,16种CTL抗原表位中,只有NAA、QKE和ERP表位发生改变,且NAA和QKE在健康人群中突变率较高。结论：山东地区EB病毒阳性淋巴瘤BRLFl基因变异类型主要为BRl-A亚型和BRl-C亚型。BRl-C亚型可能与BL相关,发生在Rta蛋白DNA结合区域和反式激活区域的突变可能对其功能产生影响,多数CTL表位序列保守提示BRLFl可以用作EBV相关淋巴瘤免疫治疗的靶基因。     

Role of the epstein-barr virus RTA protein in activation of distinct classes of viral lytic cycle genes

Initiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other's expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta's role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta's capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta's potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.