Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster R G
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA.
Proc Natl Acad Sci U S A. 2000 May 23;97(11):6108-13. doi: 10.1073/pnas.100133697.
We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I-pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 x 10(5) and 2 x 10(7) infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I-pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.
我们开发了一种八质粒DNA转染系统，用于从克隆的cDNA中拯救感染性甲型流感病毒。在这个基于质粒的表达系统中，病毒cDNA插入到RNA聚合酶I（pol I）启动子和终止子序列之间。整个pol I转录单元两侧是RNA聚合酶II（pol II）启动子和聚腺苷酸化位点。两个转录单元的方向允许从一个病毒cDNA模板合成负义病毒RNA和正义mRNA。这个pol I-pol II系统从两种细胞RNA聚合酶从其自身启动子开始转录起始，推测是在细胞核的不同区域。细胞和病毒转录及翻译机制产生的所有分子相互作用，导致产生感染性甲型流感病毒。两种甲型流感病毒A/WSN/33（H1N1）和A/Teal/HK/W312/97（H6N1）的拯救证明了该系统的实用性。将八个表达质粒转染到共培养的293T和MDCK细胞中72小时后，转染细胞上清液中的病毒产量为每毫升2×10⁵至2×10⁷个感染性病毒。我们还使用这个八质粒系统在A/Teal/HK/W312/97（H6N1）和A/WSN/33（H1N1）之间产生单重和四重重配病毒。由于pol I-pol II系统有助于设计和拯救重组及重配甲型流感病毒，它也可能适用于完全从克隆的cDNA中拯救其他RNA病毒。
