An Improved, Dual-Direction, Promoter-Driven, Reverse Genetics System for the Infectious Bursal Disease Virus (IBDV).
An Improved, Dual-Direction, Promoter-Driven, Reverse Genetics System for the Infectious Bursal Disease Virus (IBDV).
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DOI：
10.3390/v14071396
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发表时间：
2022-06-27
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传染性法氏囊病病毒（Infectious Bursal Disease Virus，IBDV）是双核糖核酸病毒科（Birnaviridae）的成员之一，它通过破坏鸡法氏囊（Bursa of Fabricius，BF）的成熟B细胞而引起雏鸡的免疫抑制。IBDV的基因组是一个双节段的双链RNA（dsRNA）。IBDV的反向遗传学系统允许通过转染的质粒DNA产生遗传操作的感染性病毒，所述质粒DNA编码两个基因组病毒RNA区段以及主要病毒蛋白。为此目的，将片段A和片段B的负义插入载体中聚合酶I启动子和相应终止子I之间。当转染到293 T细胞中时，这些质粒促进病毒负义基因组的转录，但复制正义基因组以及病毒蛋白翻译取决于VP 1和VP 3的活性。为了进一步提高拯救效率，基于pCDNA3.0载体骨架中反向的聚合酶II启动子产生双向启动子。因此，聚合酶I启动子正向转录病毒负义基因组，聚合酶II启动子转录病毒mRNA，翻译成产生感染性IBDV的病毒蛋白。我们还发现，双质粒转染的拯救效率明显高于四质粒转染。此外，该双向启动子拯救系统用于产生R186 A突变体IBDV，因为Arg 186是通过LC-MS鉴定的精氨酸单体甲基化位点。我们的数据进一步表明，Arg 186单体甲基化突变是由于VP 1聚合酶活性以及病毒复制的降低，这表明Arg 186甲基化位点是IBDV复制所必需的。
The infectious bursal disease virus (IBDV), one member of the Birnaviridae family, causes immunosuppression in young chickens by damaging the mature B cells of the bursa of Fabricius (BF), the central immune system of young chickens. The genome of IBDV is a bisegmented, double-strand RNA (dsRNA). Reverse genetics systems for IBDV allow the generation of genetically manipulated infectious virus via transfected plasmid DNA, encoding the two genomic viral RNA segments as well as major viral proteins. For this purpose, the minus-sense of both segment A and segment B are inserted into vectors between the polymerase I promoter and the corresponding terminator I. These plasmids facilitate the transcription of the viral minus-sense genome but copy the plus-sense genome as well viral protein translation depends on the activity of VP1 and VP3, when transfected into 293T cells. To further improve rescue efficiency, dual-direction promoters were generated based on the polymerase II promoter in the reverse direction in the backbone of the pCDNA3.0 vector. Therefore, the polymerase I promoter transcribes the viral minus-sense genome in the forward direction and the polymerase II promoter transcribes viral mRNA, translated into viral proteins that produce infectious IBDV. We also found that the rescue efficiency of transfecting two plasmids is significantly higher than that of transfecting four plasmids. In addition, this dual-direction promoter rescue system was used to generate R186A mutant IBDV since Arg186 is the arginine monomer-methylation site identified by LC–MS. Our data furtherly showed that the Arg186 monomer methylation mutant was due to a reduction in VP1 polymerase activity as well as virus replication, suggesting that the Arg186 methylation site is essential for IBDV replication.
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