Unité de Virologie et Immunologie Moléculaires, INRAE, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
Biological Adaptation and Ageing (B2A), Institut de Biologie Paris-Seine (IBPS), Sorbonne Université, CNRS, 75005 Paris, France.
Viruses. 2022 Mar 3;14(3):518. doi: 10.3390/v14030518.
Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.
流感病毒转录是由病毒 RNA 聚合酶（FluPol）通过帽抢夺活性催化的。FluPol 通过与 RNA 聚合酶 II（Pol II）的 RPB1 亚基的柔性 C 末端结构域（CTD）结合，从而抢先抢夺细胞 mRNA 的帽。为了更好地理解 FluPol 如何使基因组 RNA 的 3'-末端与宿主来源的引物接近，我们假设 FluPol 可能识别额外的 Pol II 亚基/结构域以确保帽抢夺。通过 Pol II 和流感 A 型 FluPol 亚基及其结构域之间的二进制互补测定，我们揭示了 PB2 的 N-第三结构域与 RPB4 之间的相互作用。通过共免疫沉淀测定证实了这种相互作用，并发现流感 B 和 C 型 FluPols 的同源结构域也存在这种相互作用。发现 RPB4 的 N-半结构域在这种相互作用中至关重要。在流感 A、B 和 C FluPols 的 PB2 的 N-第三结构域中，在保守位置生成的点状突变体表现出强烈的转录活性缺陷。这些结果表明，FluPol 与 Pol II 的几个结构域相互作用（通过 CTD 结合 Pol II），启动宿主转录，然后在 RPB4 上进行第二次转录，将 FluPol 定位在新生宿主 mRNA 的 5'-末端附近。
