Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

Influenza A virus double-helical ribonucleoprotein complex (RNP) performs transcription and replication of viral genomic RNA (vRNA). Although RNP formation occurs in the nuclei of virus-infected cells, the nuclear domains involved in this process remain unclear. Here, we show that the nucleolus is an essential site for functional RNP formation. Viral nucleoprotein (NP), a major RNP component, temporarily localized to the nucleoli of virus-infected cells. Mutations in a nucleolar localization signal (NoLS) on NP abolished double-helical RNP formation, resulting in a loss of viral RNA synthesis ability, whereas ectopic fusion of the NoLS enabled the NP mutant to form functional double-helical RNPs. Furthermore, nucleolar disruption of virus-infected cells inhibited NP assembly into double-helical RNPs, resulting in decreased viral RNA synthesis. Collectively, our findings demonstrate that NP migration into the nucleolus is a critical step for functional RNP formation, showing the importance of the nucleolus in the influenza virus life cycle. IMPORTANCE Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process. Here, we reveal by using several microscopic techniques that its major component, viral nucleoprotein (NP), temporally stays in the nucleolus, the assembly site of ribosomal RNAs/proteins, and that the formation is dependent on a nucleolar localization signal in NP. We also show that nucleolar disruption causes abortive RNP formation, resulting in a significant reduction in virus replication. Our findings demonstrate the importance of the nucleolus as the site of RNP formation and for virus replication.