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

Abstract

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.

Influenza Virus Adaptation PB2-627K Modulates Nucleocapsid Inhibition by the Pathogen Sensor RIG-I

“…It must be remarked that in all our experiments, virus stocks were used which were free of defective interfering particles (data not shown), a known activator of RIG-I (Baum et al, 2010). Moreover, in agreement with a previous study (Killip et al, 2014) we noted that addition of ActD to the mix containing CHX and LMB results in occasional background activation of antiviral signaling in uninfected cells (data not shown). We therefore conducted the majority of subsequent experiments with CHX/LMB treatment and within the short, 1 h-period of synchronized infection, conditions that were optimal for robustly studying interactions of RIG-I with incoming nucleocapsids.…”

“…It was previously stated that IFN induction by FLUAV occurs exclusively through RNA synthesis products (Killip et al, 2014; Osterlund et al, 2012). However, we observed IRF3 activation by incoming nucleocapsids even when viral transcription was shut off by ActD treatment.…”

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

“…In addition to the full-length and DI genome in the form of vRNP, recent attempts to identify authentic RIG-I ligands during IAV infection have raised the possibility that some aberrant viral RNA species contribute to IFN induction (63). Although the nature of these RNA species remains to be determined, it is plausible that some of these RNA species may have panhandle-forming potential.…”

Standing on three legs: antiviral activities of RIG-I against influenza viruses

“…These structures possess long, perfect dsRNA stretches with a 5 0 ppp and thus represent activators of RIG-I that are most likely superior to the 'original', only partially double-stranded, panhandle [19]. Therefore, viral RNA synthesis seems the dominant trigger for IFN induction once FLUAV infection is fully ongoing [21].…”