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The NF-κB signaling pathway has previously been shown to be required for efficient influenza A virus replication, although the molecular mechanism is not well understood. In this study, we identified a specific step of the influenza virus life cycle that is influenced by NF-κB signaling by using two known NF-κB inhibitors and a variety of influenza virus-specific assays. The results of time course experiments suggest that the NF-κB inhibitors Bay11-7082 and ammonium pyrrolidinedithiocarbamate inhibited an early postentry step of viral infection, but they did not appear to affect the nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. Instead, we found that the levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by the inhibitors in virus-infected cells, indicating that NF-κB signaling is intimately involved in the vRNA synthesis. Furthermore, we …