Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal
Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal
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DOI：
10.1016/j.micinf.2006.01.010
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发表时间：
2006-05-01
影响因子：
5.8
通讯作者：
Tomonaga, Keizo
中科院分区：
文献类型：
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作者：
Yanai, Hideyuki;Hayashi, Yohei;Tomonaga, Keizo
博尔纳病病毒（Borna disease virus，BDV）是一种非细胞溶解性、嗜神经性RNA病毒，在感染细胞的细胞核中复制和转录。因此，BDV RNA在细胞核中的有效合成对于该病毒的反向遗传学系统的开发至关重要。在这里，我们报告这样一个系统的发展，使用RNA聚合酶II（Pol II）启动子。BDV小基因组cDNA侧翼为锤头状核酶和丁型肝炎核酶序列，并插入Pol II启动子下游。为了提高微型基因组表达的功效，我们估计了微型基因组构建体内的几个信号序列的影响。我们发现，插入的SV40核输入序列的Pol II构建体显着增强了复制的微型基因组，即使在细胞缺乏的SV40大T抗原。这种新的系统理论上适用于任何哺乳动物细胞系，并且对于分析BDV复制和生产中的宿主或细胞类型依赖性差异将是有价值的。我们可以在此证明病毒蛋白X对BDV聚合酶活性的细胞类型依赖性抑制作用。该系统不仅可用于BDV的研究，也可用于其它负义RNA病毒的研究。(c)2006年，Elsevier SAS。All rights reserved.
Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses. (c) 2006 Elsevier SAS. All rights reserved.