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.