RNA病毒使用RNA依赖性RNA聚合酶(RdRps)来复制和转录它们的RNA基因组1。他们采用封闭的,右撇子褶皱具有保守的子域,称为棕榈,手指,和拇指1,2。保守的RdRp基序A-F协调病毒RNA模板,NTPs,和镁离子促进核苷酸缩合1。为了启动RNA合成,大多数RdRps使用引物依赖性或从头机制。而甲型流感病毒RdRp,使用加帽的RNA寡核苷酸来启动转录,以及用于复制的终端和内部从头启动的组合4。要了解甲型流感病毒RdRp如何协调这些过程,我们使用启动分析了拇指子域β发夹的功能,伸长率,和单分子FRET测定。我们的数据表明,这种β发夹对于复制过程中的末端启动至关重要,但辅助内部启动和转录。对β-发夹尖端中的单个残基的分析表明,PB1脯氨酸651对于体外和细胞培养中的有效RNA合成至关重要。总的来说,这项工作推进了我们对甲型流感病毒RNA合成的理解,并确定了病毒复制的起始平台.
RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1. They adopt a closed, right-handed fold with conserved subdomains called palm, fingers, and thumb1,2. Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs, and magnesium ions to facilitate nucleotide condensation1. For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The Influenza A virus RdRp in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the Influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation, and single-molecule FRET assays. Our data shows that this β-hairpin is essential for terminal initiation during replication, but auxiliary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of Influenza A virus RNA synthesis and identifies the initiation platform of viral replication.