Burysek L, Yeow W S, Pitha P M
Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
J Hum Virol. 1999 Jan-Feb;2(1):19-32.
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.
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.
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.
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.
人类疱疹病毒8型/卡波西肉瘤疱疹病毒（HHV-8/KSHV）除了含有病毒复制所需的基因外，还拥有一组独特的非结构基因，这些基因可能是病毒模拟的一部分，并有助于病毒在体内的复制和发病机制。其中，HHV-8编码四个开放阅读框（ORF），它们与干扰素调节因子（IRF）家族的转录因子具有同源性。在本研究中，我们证明这些ORF之一（vIRF-2）编码一种分子量为18kd的蛋白质，其表达模式和特性与细胞IRF以及先前鉴定的vIRF-1不同。
我们通过聚合酶链反应（PCR）克隆了vIRF-2，并通过Northern印迹和逆转录-聚合酶链反应（RT-PCR）研究其表达。通过氯霉素乙酰转移酶（CAT）测定法在瞬时转染的哺乳动物细胞中测试其生物学活性。我们通过电泳迁移率变动分析（EMSA）表征其DNA结合特异性，并通过体外下拉测定法表征其蛋白质-蛋白质相互作用。
虽然在HHV-8阳性的BCBL-1肿瘤细胞系中可检测到低水平的vIRF-2 mRNA，但12-O-十四酰佛波醇-13-乙酸酯（TPA）处理并不会与主要裂解周期基因一起刺激vIRF-2基因的表达。能够形成同源二聚体的重组vIRF-2不会特异性结合与干扰素刺激反应元件（ISRE）对应的寡脱氧核苷酸重复序列，但它会结合NF-κB结合位点。由vIRF-2和RelA（p65）激活域产生的融合蛋白刺激含有两个NF-κB位点的HIV LTR的转录活性，但不会刺激仅含有一个NF-κB位点的干扰素-β（IFNB）启动子。通过体外结合测定检测到重组vIRF-2与细胞IRF如IRF-1、IRF-2和ICSBP之间的相互作用，但未发现IRF-3与vIRF-2之间的相互作用。还观察到vIRF-2与RelA（p65）和p300的羧基末端部分之间的相互作用。在瞬时转染测定中，vIRF-2抑制感染细胞中IRF-1或IRF-3介导的干扰素-α（IFNA）基因启动子的转录激活，并下调RelA（p65）刺激的HIV LTR活性。
这些结果表明，通过与细胞转录因子和辅因子相互作用，vIRF-2可能调节早期炎症基因的表达，并可能使免疫系统失调。
