Structural study of Polymerase (PA subunit endo mutant E119D) of influenza H7N9 virus purified in high five cells

The influenza virus polymerase (FluPol) is implicated in both transcription and replication of the viral RNA (vRNA).Transcription enables the production of an mRNA which is utilised by the host cell to generate viral proteins. These viral proteins are required for virus replication and assembly of progeny virions. In order to initiate transcription FluPol must perform 'cap-snatching'. The FluPol cap-binding domain binds to a 5' capped mRNA primer and the endonuclease cleaves the cap from the rest of the mRNA strand. Our aim is to capture FluPol during 'cap-snatching' using cryo-EM. In order to achieve this we have combined a H7N9 FluPol mutant which has reduced endonuclease activity with a long capped mRNA primer, thereby stalling FluPol during the process of 'cap-snatching'.

流感病毒聚合酶（influenza virus polymerase，FluPol）参与病毒RNA（viral RNA，vRNA）的转录与复制过程。转录过程可产生信使RNA（messenger RNA，mRNA），宿主细胞可利用该mRNA合成病毒蛋白。这些病毒蛋白是病毒复制与子代病毒粒子组装过程所必需的。为启动转录，FluPol必须执行“帽抢取（cap-snatching）”过程。FluPol的帽结合结构域可结合带有5'帽结构的mRNA引物，而核酸内切酶会将mRNA链其余部分的帽结构切除。本研究的目标是利用冷冻电镜（cryo-electron microscopy，cryo-EM）在“帽抢取”过程中捕获FluPol。为达成这一目标，我们将核酸内切酶活性降低的H7N9型FluPol突变体与长链带帽mRNA引物相结合，从而使FluPol在“帽抢取”过程中停滞。