Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: Functional analysis and transcriptional modulation
Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: Functional analysis and transcriptional modulation
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DOI：
10.1016/j.molimm.2008.08.265
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发表时间：
2008-12-01
影响因子：
3.6
通讯作者：
Secombes, C. J.
中科院分区：
文献类型：
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作者：
Holland, J. W.;Bird, S.;Secombes, C. J.
已知哺乳动物中的干扰素调节因子（IRF）3和7作为与IFN和干扰素刺激基因（ISG）内的IRF结合元件（IRF-Es）和干扰素刺激应答元件（ISRE）结合的转录复合物的一部分，在调节I型干扰素（IFN）对病毒感染的应答中至关重要。在这里，我们报告的全长cDNA同源的虹鳟鱼（RT）IRF 7和IRF 3的测序和表征，首次在鱼类。RtIRF 3基因全长2127 bp，5 '-UTR上游有159 bp的AUG，3'-UTR上游有573 bp。发现RtIRF 7为2055 bp，具有102 bp的5 '-UTR和705 bp的3'-UTR。开放阅读框（ORF）分别翻译成464个氨基酸和415个氨基酸的蛋白质，每个蛋白质具有含有色氨酸簇的推定DNA结合结构域（DBD），这是所有IRF家族成员的特征。存在推定的IRF关联结构域（IAD），富含丝氨酸的C末端结构域（在鳟鱼IRF 3中保守性较差），和系统发育分析将这两个基因定位在IRF 3亚家族中。在鳟鱼巨噬细胞系RTS-11中，发现两种基因均被poly I：C、I型重组虹鳟（r）IFN（第二同种型，I型rIFN）、II型rIFN（rIFN γ）、LPS和rIL-1 β上调。Poly I：C和I型rIFN也诱导鳟鱼成纤维细胞系（RTG-2）中的IRF 3和IRF 7表达。RTG-2细胞与每个IRF融合到GFP的瞬时转染揭示了主要的细胞质分布，发现最强烈的细胞核周围，并在较小程度上，在细胞核内。在Mx-1-荧光素酶报告细胞系RTG-P1中瞬时转染rtIRF 3，显示相对于载体对照，荧光素酶活性适度增加，其在过表达DBD截短形式的rtIRF 3的细胞中丧失。相对于对照，rtIRF 7的全长和DBD截短形式都增加了报告活性，尽管程度不显著。电迁移率变动分析（EMSA）并没有揭示一个特定的相互作用，每个IRF和ISRE元素中发现的Mx-1启动子，虽然Mx-1 ISRE结合特异性内源性转录复合物。这些数据支持rtIRF 3和rtIRF 7是调节鱼类抗病毒应答的重要分子的前提，rIFN γ对rtIRF 3/7表达的影响意味着这些IRFs在免疫过程中的作用，而不是1型IFN驱动的抗病毒应答。(c)2008年由Elsevier Ltd.出版
Interferon regulatory factors (IRF) 3 and 7 in mammals are known to be crucial in regulating the type I interferon (IFN) response to viral infection as part of transcriptional complexes binding to IRF-binding elements (IRF-Es) and interferon stimulatory response elements (ISREs) within IFN and interferon-stimulated genes (ISGs). Here we report the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt)IRF7 and, for the first time in fish, IRF3. RtIRF3 consists of 2127 bp with a 159 bp 5'-UTR-containing two upstream AUGs and a 573 bp 3'-UTR. RtIRF7 was found to be 2055 bp, with a 102 bp 5'-UTR and a 705 bp 3'-UTR. The open reading frames (ORFs) translate into 464 amino acid and 415 amino acid proteins, respectively, each possessing a putative DNA-binding domain (DBD) containing a tryptophan cluster, which is characteristic of all IRF family members. The presence of putative IRF association domain (IAD)s, serine-rich C terminal domains (poorly conserved in trout IRF3), and phylogenetic analysis places the two genes in the IRF3 subfamily. Both genes were found to be upregulated by poly I:C, type I recombinant rainbow trout (r) IFN (second isoform, type I rIFN), type II rIFN (rIFN gamma), LPS, and rIL-1 beta in the trout macrophage cell line, RTS-11. Poly I:C and type I rIFN also induced IRF3 and IRF7 expression in a trout fibroblast cell line (RTG-2). Transient transfection of RTG-2 cells with each IRF fused to GFP revealed a predominant cytoplasmic distribution found most intensely around the nucleus and, to a lesser extent, within cell nuclei. Transient transfection of rtlRF3 in the Mx-1-luciferase reporter cell line, RTG-P1, revealed a modest increase in luciferase activity relative to the vehicle control, which was lost in cells over-expressing a DBD-truncated form of rtIRF3. Both full-length and DBD-truncated forms of rtIRF7 increased reporter activity relative to the control, although to a non-significant extent. Electromobility shift assays (EMSAs) did not reveal a specific interaction between each IRF and the ISRE element found in the Mx-1 promoter, although the Mx-1 ISRE bound specifically to endogenous transcriptional complexes. These data support the premise that rtIRF3 and rtIRF7 are important molecules in the regulation of antiviral responses in fish, with the impact of rIFN gamma on rtIRF3/7 expression implying a role for these IRFs in immune processes other than type 1 IFN-driven antiviral responses. (c) 2008 Published by Elsevier Ltd.