Activation of the Interferon Induction Cascade by Influenza A Viruses Requires Viral RNA Synthesis and Nuclear Export

We have examined the requirements for virus transcription and replication and thus the roles of input and progeny genomes in the generation of interferon (IFN)-inducing pathogen-associated molecular patterns (PAMPs) by influenza A viruses using inhibitors of these processes. Using IFN regulatory factor 3 (IRF3) phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-β promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a variety of influenza A viruses in the presence of cycloheximide or nucleoprotein (NP) small interfering RNA (siRNA), which inhibits viral protein synthesis and thus complementary ribonucleoprotein (cRNP) and progeny viral RNP (vRNP) synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. While the exact nature of the viral PAMPs remains to be determined, our data suggest that in this experimental system the major influenza A virus PAMPs are distinct from those of incoming genomes or progeny vRNPs. IMPORTANCE The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection, and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer(s) still remains to be identified.