Carla Gallardo Flores, Queen's University

Carla E. Gallardo Flores, Lindsay Yu, Elana Kertzman, Che C. Colpitts - Department of Biomedical and Molecular SciencesQueen's University

Suppressing & Conquering

Protein kinase R (PKR) is an innate immune sensor that is activated by double-stranded RNA (dsRNA), leading to the inhibition of eukaryotic translation initiation factor 2A and causing a halt in protein translation and the formation of stress granules (SG). Hepatitis C virus (HCV) non-structural protein 5A (NS5A) has been reported to inhibit PKR. However, PKR is still activated in cells infected with HCV JFH-1. To address this discrepancy, we sought to characterize the potential mechanism(s) of PKR antagonism by NS5A, by comparing NS5A from different HCV strains (J4 and JFH-1) with known viral PKR antagonists, such as poxvirus K3L, in transfection experiments. To determine whether NS5A can directly inhibit PKR activation, we employed a GyrB-PKR construct that is selectively dimerized and activated by coumermycin. Coumermycin treatment led to SG formation and decreased protein synthesis in 293T cells. However, neither NS5A-JFH1 nor NS5A-J4 inhibited GyrB-PKR activation, while K3L, a pseudo-substrate inhibitor of PKR, blocked PKR signaling. We next transfected cells with poly(I:C), a dsRNA mimic, to test whether NS5A inhibits dsRNA-induced PKR activation. Neither NS5A-JFH1 nor NS5A-J4 inhibited PKR activation (autophosphorylation), SG formation, or protein translation. Doxycycline-inducible expression of NS5A in Huh7.5.1 hepatoma cells confirmed that NS5A does not inhibit activation of PKR by dsRNA. We are currently investigating whether NS5A inhibits PKR activation by the 5′-UTR or 3′-UTR regions of HCV RNA, which could activate PKR via stem-loop structures and are known to bind NS5A. Overall, our findings suggest that HCV NS5A is not an effective PKR inhibitor.