Ramamoorthy S, Huang F F, Huang Y W, Meng X J
Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913, USA.
Virus Res. 2009 Nov;145(2):236-43. doi: 10.1016/j.virusres.2009.07.009. Epub 2009 Jul 21.
Porcine circovirus type 2 (PCV2) is an economically important swine pathogen. It has been shown that treatment of PCV2-infected cells with interferon gamma (INF-gamma) or alpha (IFN-alpha) increases PCV2 replication in vitro, and that an interferon-stimulated response element (ISRE)-like sequence was identified in the PCV2 genome. To determine if the ISRE is involved in viral response to IFNs, several ISRE mutants of PCV2 were created by serial mutations of the ISRE sequence. Treatment of ISRE mutants-infected cells with IFN-gamma or IFN-alpha showed a progressive diminishment in the enhanced viral replication in correlation with increasing alterations to the ISRE sequence. To determine if the ISRE was necessary and sufficient for IFN-mediated enhancement of PCV2 replication, DNA fragments spanning the ISRE-containing promoter region of the rep gene from wildtype and ISRE-mutant PCV2 were tested in a luciferase reporter gene system. 3D4/31 cells transfected with reporter gene constructs were treated with IFN-gamma or IFN-alpha, respectively. The results showed that the untreated controls for both ISRE-mutant and wildtype PCV2 had higher levels of luciferase reporter activity than IFN-treated samples, indicating that, when removed from the context of whole viral genome, the ISRE-like activity of the sequence was lost. Furthermore, treatment with IFNs diminished the promoter activity regardless of the mutation, suggesting that other cis elements in the viral genome may be required for regulating the ISRE-mediated gene transcription. In conclusion, the PCV2 ISRE, when present in the context of intact virus but not in isolation, influences the interferon-mediated enhancement of PCV2 replication in vitro.
猪圆环病毒2型（PCV2）是一种具有重要经济意义的猪病原体。研究表明，用γ干扰素（INF-γ）或α干扰素（IFN-α）处理PCV2感染的细胞会增加PCV2在体外的复制，并且在PCV2基因组中鉴定出了一个类似干扰素刺激反应元件（ISRE）的序列。为了确定ISRE是否参与病毒对干扰素的反应，通过对ISRE序列进行系列突变，构建了几个PCV2的ISRE突变体。用IFN-γ或IFN-α处理ISRE突变体感染的细胞，结果显示，随着ISRE序列改变的增加，病毒复制增强的现象逐渐减弱。为了确定ISRE对于干扰素介导的PCV2复制增强是否必要且充分，在荧光素酶报告基因系统中测试了来自野生型和ISRE突变型PCV2的包含rep基因ISRE的启动子区域的DNA片段。分别用IFN-γ或IFN-α处理转染了报告基因构建体的3D4/31细胞。结果表明，ISRE突变型和野生型PCV2的未处理对照的荧光素酶报告活性水平均高于干扰素处理的样品，这表明当从整个病毒基因组中去除该序列时，其类似ISRE的活性丧失。此外，无论是否存在突变，干扰素处理都会降低启动子活性，这表明病毒基因组中的其他顺式元件可能是调节ISRE介导的基因转录所必需的。总之，PCV2的ISRE在完整病毒存在的情况下而非单独存在时，会影响体外干扰素介导的PCV2复制增强。
