Date Published: March 3, 2016
Publisher: Public Library of Science
Author(s): Alissa M. Pham, Felicia Gilfoy Santa Maria, Tanaya Lahiri, Eugene Friedman, Isabelle J. Marié, David E. Levy, Charles E Samuel.
Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.
The innate immune response allows for the rapid production of type I interferons (IFNs) and other proinflammatory cytokines to counteract invading viral pathogens. This response relies, in part, on a group of molecules collectively referred to as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns generated during the course of infection. The detection of virus infection is mediated primarily by cytoplasmic sensors for both RNA and DNA, which include members of the RIG-like helicase (RLH) family for RNA detection and a variety of cytoplasmic proteins for detection of DNA .
PKR is a key component of the antiviral response to a wide variety of viruses [69, 70]. This antiviral effect has been largely attributed to its well-characterized ability to phosphorylate and, thereby, inactivate eIF2α, leading to inhibition of translation of both viral and cellular RNA. As a consequence of eIF2α phosphorylation and the subsequent inhibition of translational initiation, PKR activation also leads to the formation of stress granules that appear to have antiviral function . Early studies indicated that PKR may also play a direct role in IFN induction in response to pIC, a dsRNA mimetic [36, 49]; however, a number of more recent genetic and biochemical studies have suggested that PKR can be superfluous for IFN induction, at least in response to some viruses, throwing into question whether PKR acts as a sensor or an effector in the antiviral response. For instance, IFN induction by pIC or Newcastle disease virus (NDV) infection was intact in PKR-null mice in vivo, and an apparent in vitro deficiency could be reversed by IFN priming, suggesting that PKR-independent pathways mediated IFN induction . Discovery of cytoplasmic dsRNA sensors, RIG-I, MDA5, and LGP2 and their associated signaling pathways, has provided a framework for understanding the primary pathways for innate immune responses to RNA viruses, without a documented role for PKR . Nonetheless, experimental data have suggested that PKR may be a component of IFN production in response to some viruses, including West Nile virus , Theiler’s murine encephalitis virus (TMEV), EMCV , measles virus  and Semliki Forest virus (SFV) . Here, we provide evidence that PKR is involved in MDA5-mediated responses to virus infection and is necessary for IFN induction. Mechanistically, we found that MDA5 and PKR proteins associate in a complex, and that MDA5 augments virus-induced activation of PKR. These results suggest that that PKR participates as part of the viral sensor machinery in concert with MDA5 to recognize foreign RNA and induce IFN.