P117 Regulation of actin dynamics by Protein Kinase R control of gelsolin enforces basal innate immune defense

Abstract

Introduction The primary resistance to pathogens is heavily reliant on the innate immune response. This resistance depends upon both basal and inducible defenses. Contrasting to inducible resistance through cytokine induction, basal defense mechanisms are much less evident. Here we detail how the antiviral effector Protein Kinase R (PKR) interacts with the key actin-regulatory protein gelsolin to regulate actin dynamics and control cytoskeletal functions under homeostatic conditions. In addition, up-regulation of PKR by Interferon can enhance this interaction. Methods The interaction was confirmed by recombinant proteins, Immunoprecipitations, FRET and bi-molecular Fluorescence (Bi-FC). Investigation of actin dynamics was performed with recombinant proteins and in cells via live cell microscopy and FRAP. Endocytosis and uptake of viral particles/bacteria was examined via live cell microscopy, 3D modelling and replicated with in vivo mouse models. Results Through this mechanism, PKR directly inhibits Gelsolin, blocking its ability to sever actin filaments and affecting actin-dependent processes that include membrane ruffling, migration, endocytosis and accordingly, PKR counteracts pathogen entry into the cell. This also impacts the direct function of cells of the immune compartment including phagocytosis and activation. Conclusion These findings identify a new layer of host resistance, with the regulation of actin-modifying proteins during the innate immune response bolstering first-line defense against intracellular pathogens and having a sustained effect on virus production and the overall consequence of infection. This response can also be enhanced by Interferon treatment to up-regulate PKR. Moreover, modulating the cell cytoskeleton with specific targets could potentially be used to elicit broad protection against pathogens.