Lubyova B, Pitha P M
Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
J Virol. 2000 Sep;74(17):8194-201. doi: 10.1128/jvi.74.17.8194-8201.2000.
The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10. 6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.
人类疱疹病毒8型（HHV-8）的基因组包含一组开放阅读框（ORF），这些开放阅读框编码的蛋白质与干扰素调节因子（IRF）家族的细胞转录因子具有同源性。先前已鉴定并对其中两个同源物vIRF-1和vIRF-2进行了功能分析。在本研究中，我们鉴定了一个新基因，命名为vIRF-3，它编码于先前预测的ORF K10.5和我们新鉴定的ORF K10.6内。用vIRF-3特异性探针从BCBL-1细胞中提取RNA进行Northern印迹分析以及逆转录-PCR分析显示，存在一个2.2 kb的单一转录本，其编码区域有一个剪接位点。经12-O-十四烷酰佛波醇-13-乙酸酯处理后，BCBL-1细胞中的vIRF-3 mRNA水平升高，其表达动力学与早期即刻基因相似。vIRF-3 ORF编码一种73 kDa的蛋白质，与细胞IRF-4以及HHV-8编码的vIRF-2和K11具有同源性。在使用IFNACAT报告基因的瞬时转染试验中，vIRF-3作为IRF-3和IRF-7的显性负性突变体发挥作用，并抑制病毒介导的IFNA启动子的转录活性。同样，在小鼠L929细胞中过表达vIRF-3会导致病毒介导的生物活性干扰素合成受到抑制。这些结果表明，通过靶向在α/β干扰素（IFN）基因激活中起关键作用的IRF-3和IRF-7，HHV-8进化出了一种机制，通过该机制它直接破坏IRF的功能，并下调作为先天免疫重要组成部分的IFN基因的诱导。
