The innate immune responses play critical roles in combating viral infection. A key feature of this response is production of type I interferons (IFNs) which induce an antiviral and anti-proliferative state in the cells. This process requires activation of transcription factors, such as nuclear factor-κB (NF-κB) and interferon regulatory factor-3 (IRF-3). It has been reported that paramyxovirus infection can activate expression of cytokines such as IFN-β, a type I IFN. However, the mechanism of the activation by virus is not well understood. The cytoplasmic viral sensors, the RNA helicases, RIG-I or MDA5, play critical roles in induction of interferon signaling through detection of specific viral components in the infected cells. However, the origin and molecular identity of these viral RNA ligands is not well characterized. Intriguingly, viruses have strategies to evade this host defense response. This interplay between innate immunity and virus infections, is not only crucial to understand virus-host relationships, but also may lead to development of novel antiviral therapeutics.

In this work, we have studied the activation and modulation of innate immune responses by viruses, focusing on the NF-κB and the interferon signaling pathways using parainfluenza virus 5 (PIV5), a prototypical paramyxovirus as our model system. Paramyxoviruses are enveloped nonsegmented negative stranded (NNS) RNA viruses that include many important human and animal pathogens. PIV5 is a poor inducer of the antiviral responses. However, a recombinant PIV5 lacking the conserved region of the V protein (rPIV5VΔC) activates expression of cytokines IFN-β and IL-6 through a NF-κB dependent pathway.

We investigated the role of viral proteins in this activation and determined that the large (L) polymerase protein of PIV5 can activate NF-κB leading to activation of IFN-β and IL-6. The L protein of PIV5 is the main component of viral RNA-dependent RNA polymerase and is about 250 kDa. It contains six domains that are conserved among all NNS RNA viruses. We analyzed the mechanism of the activation of NF-κB by the L protein and found that AKT1, a serine/threonine kinase plays a critical role in activation of NF-κB by L. We found that the L protein interacts with AKT1 and enhances its phosphorylation. We further mapped the region within the L that is sufficient to activate NF-κB to domain II and discovered its role in activation of IFN-β. Surprisingly, we found that the RNA transcribed from domain II of the L gene is sufficient to activate NF-κB in an AKT independent pathway and can also induce interferon expression. We have further evaluated the mechanism of this activation and have determined that this viral RNA activates IFN-β through MDA5 and endoribonuclease, RNase L dependent pathway.

Our study has revealed novel roles of the L gene in the host signaling pathways. Our work is the first report identifying a natural ligand for MDA5 that can induce interferon expression and can serve as a possible prototype for the development of broad antiviral therapeutics.