A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region
A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region
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DOI：
10.1128/jvi.05554-11
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发表时间：
2011-12-01
影响因子：
5.4
通讯作者：
Tomonaga, Keizo
中科院分区：
文献类型：
--
作者：
Daito, Takuji;Fujino, Kan;Tomonaga, Keizo
博尔纳病病毒（Borna disease virus，BDV）是一种不分节段的负链RNA病毒，感染多种哺乳动物物种，并容易在脑细胞中建立持久的持续感染。因此，这种病毒可能是一个有前途的候选人作为一种新的RNA病毒载体，使基因在中枢神经系统（CNS）的稳定表达。以前的研究表明，基因组的5'非翻译区是外源基因插入和表达的唯一位点。在这项研究中，我们建立了一个新的BDV载体，其中一个额外的转录盒已插入到一个顺反子间的非编码区之间的病毒磷蛋白（P）和基质（M）基因。在P和M基因之间携带绿色荧光蛋白（GFP）的重组BDV（rBDV），rBDV P/M-GFP，在培养的细胞和啮齿动物脑中有效地表达GFP长时间而没有衰减。此外，我们通过G基因的瞬时或稳定表达的反式互补系统产生了非繁殖rBDV，Delta GLLP/M，其缺乏包膜糖蛋白（G）和聚合酶基因（L）内的剪接内含子。有趣的是，rBDV Delta GLLP/M在缺乏G.使用持续感染的rBDV Delta GLLP/M感染的细胞，我们确定了BDV G的胞质尾区（CT）中的氨基酸区域，该区域对于感染性rBDV颗粒的释放是重要的，并且还证明了CT区域对于具有水泡性口炎病毒G蛋白的假型rBDV的产生可能是关键的。我们的研究结果表明，新建立的BDV载体构成了一个替代的工具，不仅在中枢神经系统中稳定表达外源基因，但也了解包膜病毒粒子的释放机制。
Borna disease virus (BDV), a nonsegmented, negative- strand RNA virus, infects a wide variety of mammalian species and readily establishes a long- lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5' untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/ M- GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, Delta GLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV Delta GLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV Delta GLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions.