title: Monitoring activation of the antiviral pattern recognition receptors RIG-I and PKR by limited protease digestion and native PAGE.
abstract: Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established. Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples. Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.
authors.affiliation: Institute for Virology, Philipps-University Marburg.
authors.forename: Michaela
authors.initials: M
authors.lastname: Weber
authors.affiliation: Institute for Virology, Philipps-University Marburg; friedemann.weber@staff.uni-marburg.de.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Weber
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D012367:RNA, Viral / Q000378:metabolism*; D011971:Receptors, Immunologic; D012296:Rift Valley fever virus / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D011971:Receptors, Immunologic; D019892:eIF-2 Kinase; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases