Dos Santos Afonso Emmanuel, Escriou Nicolas, Leclercq India, van der Werf Sylvie, Naffakh Nadia
Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 PARIS Cedex 15, France.
Virology. 2005 Oct 10;341(1):34-46. doi: 10.1016/j.virol.2005.06.040.
We generated recombinant A/WSN/33 influenza A viruses expressing a PB2 protein fused to a Flag epitope at the N- (Flag-PB2) or C-terminus (PB2-Flag), which replicated efficiently and proved to be stable upon serial passage in vitro on MDCK cells. Rescue of PB2-Flag viruses required that the 5' end of the PB2 segment was kept identical to the wild-type beyond the 34 noncoding terminal nucleotides. This feature was achieved by a duplication of the 109 last nucleotides encoding PB2 between the Flag sequence and the 5'NCR. In PB2 mini-genomes rescue experiments, both the 5' and 3' coding ends of the PB2 segment were found to promote the incorporation of mini-genomes into virions. However, the presence of the Flag sequence at the junction between the 3'NCR and the coding sequence did not prevent the rescue of Flag-PB2 viruses. Our observations define requirements that may be useful for the purpose of engineering influenza RNAs.
我们构建了重组A/WSN/33甲型流感病毒，其表达在N端（Flag-PB2）或C端（PB2-Flag）与Flag表位融合的PB2蛋白，这些病毒能高效复制，并且在体外MDCK细胞上连续传代后证明是稳定的。拯救PB2-Flag病毒要求PB2片段的5'端在34个非编码末端核苷酸之后与野生型保持一致。这一特性是通过在Flag序列和5'NCR之间重复编码PB2的最后109个核苷酸来实现的。在PB2微型基因组拯救实验中，发现PB2片段的5'和3'编码末端均能促进微型基因组掺入病毒粒子。然而，在3'NCR与编码序列的连接处存在Flag序列并不妨碍拯救Flag-PB2病毒。我们的观察结果确定了一些要求，这些要求可能对工程化流感RNA有用。
