Unique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2).
Unique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2).
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发表时间：
1999
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通讯作者：
L. Burýšek;Yeow Ws;Pitha Pm
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作者：
L. Burýšek;Yeow Ws;Pitha Pm
目的人类疱疹病毒8型/卡波西肉瘤疱疹病毒(HHV-8/KSHV)除了病毒复制所需的基因外，还含有一组独特的非结构基因，这些基因可能是病毒模仿的一部分，在体内参与病毒复制和致病。其中，HHV-8编码四个开放阅读框(ORF)，与干扰素调节因子(IRF)家族的转录因子具有同源性。在本研究中，我们证明了其中一个开放阅读框(vIRF-2)编码了一个迁移率为18kd的蛋白质，其表达模式和性质与细胞内的iRFs和先前鉴定的vIRF-1不同。方法采用聚合酶链式反应(PCR)技术克隆vIRF-2基因，用Northern印迹和逆转录聚合酶链式反应(RT-PCR)检测其表达。用氯霉素乙酰转移酶(CAT)比色法检测瞬时转基因哺乳动物细胞的生物学活性。我们用凝胶迁移率改变分析(EMSA)和体外下拉实验研究了它的DNA结合特异性和蛋白质与蛋白质的相互作用。结果虽然在HHV-8阳性的BCBL-1肿瘤细胞系中检测到低水平的vIRF-2mRNAs，但12-0-十四酰佛波醇-13-乙酸酯(TPA)处理不能刺激vIRF-2基因和原裂解周期基因的表达。重组vIRF-2可形成同源二聚体，不与干扰素刺激反应元件(ISRE)对应的寡脱氧核苷酸重复序列特异结合，但确实与核因子-kappa B结合位点结合。由vIRF-2和relA(P65)激活结构域产生的融合蛋白刺激含有两个NF-kappa B位点的HIV LTR的转录活性，但不刺激只含有一个NF-kappa B位点的干扰素-β(IFNB)启动子。体外结合实验检测到重组vIRF-2与IRF-1、IRF-2、ICSBP等细胞内IRF相互作用，但未发现IRF-3与VIRF-2相互作用。还观察到vIRF-2与relA(P65)和p300的羧基末端部分的相互作用。在瞬时转染实验中，vIRF-2抑制了IRF-1或IRF-3介导的感染细胞中干扰素-α(IFNA)基因启动子的转录激活，并下调了RelA(P65)刺激的HIV LTR的活性。结论vIRF-2可能通过与细胞转录因子和辅因子相互作用，调节早期炎症基因的表达，并有可能解除免疫系统的调节。
OBJECTIVE Human herpesvirus 8/Kaposi's sarcoma herpesvirus (HHV-8/KSHV) contains, in addition to genes required for viral replication, an unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that show homology to the transcription factors of the interferon regulatory factor (IRF) family. In this study we demonstrate that one of these ORFs (vIRF-2) encodes a protein with mobility of 18 kd which has distinct pattern of expression and properties from the cellular IRFs and the previously characterized vIRF-1. METHODS We cloned vIRF-2 by polymerase chain reaction (PCR) and studied its expression by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Biologic activities were tested by chloramphenicol acetyltransferase (CAT) assay in transiently transfected mammalian cells. We characterized its DNA binding specificity by electrophoretic mobility shift analysis (EMSA) and its protein-protein interactions by in vitro pull-down assay. RESULTS Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-positive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primary lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does not bind specifically to the oligodeoxynucleotide repeats corresponding to the interferon-stimulated response element (ISRE), but it does bind to the NF-kappa B binding site. The fusion protein generated from vIRF-2 and the RelA (p65) activation domain stimulates transcriptional activity of HIV LTR, which contains two NF-kappa B sites, but does not stimulate the interferon-beta (IFNB) promoter, which contains only one NF-kappa B site. Interaction between recombinant vIRF-2 and cellular IRFs such as IRF-1, IRF-2, and ICSBP was detected by in vitro binding assay, but no interaction between IRF-3 and vIRF-2 was found. Interaction of vIRF-2 with RelA (p65) and the carboxy-terminal part of p300 was also observed. In a transient transfection assay, vIRF-2 inhibits the IRF-1- or IRF-3-mediated transcriptional activation of interferon-alpha (IFNA) gene promoter in infected cells and downmodulates RelA (p65)-stimulated activity of HIV LTR. CONCLUSIONS These results suggest that, by interacting with cellular transcription factors and cofactors, vIRF-2 may modulate the expression of the early inflammatory genes and potentially deregulate the immune system.