A reverse-genetics system for Influenza A virus using T7 RNA polymerase
A reverse-genetics system for Influenza A virus using T7 RNA polymerase
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DOI：
10.1099/vir.0.82452-0
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发表时间：
2007-04-01
影响因子：
3.8
通讯作者：
Fouchier, Ron A. M.
中科院分区：
文献类型：
--
作者：
de Wit, Emmie;Spronken, Monique I. J.;Fouchier, Ron A. M.
目前可用的甲型流感病毒的反向遗传学系统都是基于RNA聚合酶1对基因组RNA的转录，但这种聚合酶的种属特异性是一个缺点。已经开发了含有T7 RNA聚合酶启动子、丁型肝炎病毒核酶序列和T7 RNA聚合酶终止子序列的反向遗传学载体。为了在微型基因组测定中实现最佳表达，确定病毒RNA应以负义方向插入该载体中，在T7 RNA聚合酶启动子下游具有两个额外的G残基。还显示，当使用具有核定位信号的T7 RNA聚合酶时，微型基因组的表达更有效。通过使用该反向遗传学系统，重组流感病毒A/PR/8/34的生产效率比使用类似的基于聚合酶I的反向遗传学系统更高。此外，流感病毒A/NL/219/03可以从293 T、MDCK和QT 6细胞中拯救出来。因此，已经开发了用于拯救甲型流感病毒的反向遗传学系统，这将有助于基础研究和在各种细胞系中生产疫苗种子株。
The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase 1, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.