Examining the Dynamic Architecture of the Influenza Virus Genome

流感病毒的组装是一个复杂的协调过程。流感病毒基因组由八个负义单链RNA片段组成，其被病毒编码的核蛋白（NP）寡聚体包被。在感染过程中，新合成的病毒RNA（vRNA）和NP在细胞核中缔合。由vRNA、NP和病毒聚合酶复合物组成的病毒核糖核蛋白（vRNP）片段被转运到质膜，在质膜上所有八个vRNP片段被包装成单个病毒体。vRNA和NP之间的相互作用不仅用于保护宿主细胞内的vRNA，而且还促进vRNP区段进出细胞核的转运、vRNA合成以及所有八个vRNA区段的选择性包装。我们最近使用下一代测序技术表明，NP结合到整个病毒基因组的选择区域，而不是像以前假设的那样均匀地结合在一起。然而，目前尚不清楚在体外对RNA没有序列特异性结合活性的NP如何定位于vRNA上的靶位点。NP在vRNA上的靶位点的这种特异性定位对于维持病毒适应性至关重要。此外，大量证据表明，vRNA片段在组装过程中在各个片段之间形成RNA-RNA相互作用，以促进完整基因组的包装。我们的目标是研究NP与vRNA的结合如何随着片段向质膜的进展而变化，以及在转运过程中RNA-RNA相互作用如何演变。该提案的结果将提供对流感vRNP结构和控制流感vRNP组装的复杂机制的更好理解。

Assembly of influenza viruses is a complex coordinated process. The influenza virus genome consists of eight negative-sense single-stranded RNA segments that are coated by virally encoded nucleoprotein (NP) oligomers. During infection, the newly synthesized viral RNA (vRNA) and NP associate in the nucleus. The viral ribonucleoprotein (vRNP) segments, composed of vRNA, NP and the viral polymerase complex, are transported to the plasma membrane, where all eight vRNP segments are packaged into a single virion. The interaction between vRNA and NP not only serves to protect vRNA inside the host cell, but also promotes transport of the vRNP segments into and out of the nucleus, vRNA synthesis, and selective packaging of all eight vRNA segments. We have recently shown using techniques entailing next-generation sequencing that NP binds to select regions throughout the viral genome and is not uniformly bound like ‘beads on a string’ as previously posited. However, it remains unclear how NP, which in vitro has no sequence-specific binding activity towards RNA, localizes to its target sites on vRNA. This specific localization of NP to its target sites on vRNA is essential to maintain viral fitness. Furthermore, a body of evidence suggests that vRNA segments form RNA-RNA interactions between individual segments during the assembly process to facilitate the packaging of the complete genome. Our objectives in this proposal are to examine how NP binding to vRNA changes as the segments progress towards the plasma membrane and how RNA-RNA interactions evolve during transit. The outcome of this proposal will provide a greater understanding of the influenza vRNP structure and the complex mechanism governing influenza vRNP assembly.