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.