Cooperative Roles of Fish Protein Kinase Containing Z-DNA Binding Domains and Double-Stranded RNA-Dependent Protein Kinase in Interferon-Mediated Antiviral Response

Abstract

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2␣ (eIF2␣). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2␣ kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2␣. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2␣ than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2␣ independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.

Zebrafish foxo3b Negatively Regulates Antiviral Response through Suppressing the Transactivity of irf3 and irf7

“…Through inducing the expression of viperin, zebrafish IFN participates in the antiviral response (50). Two typical IFN-inducible genes, pkr and pkz, can suppress replication of grass carp reovirus (51,52). To date, zebrafish have been widely employed to investigate the function of genes involved in innate immunity (43,(53)(54)(55).…”

Fish MAVS is involved in RLR pathway-mediated IFN response

“…No LPS contamination in recombinant IFN was verified by a cooking experiment where the cooked rIFN lose its ability to induce downstream ISG expression. Transfection assays were performed according to previous reports [23,24,30]. In brief, CAB cells seeded in 24-well or 6-well plates overnight were treated with the indicated plasmids (each 0.25 mg in 24-well plates or 1.6 mg in 6-well plates) and 1 ml or 10 ml FuGENE ® HD Transfection Reagent (Roche) in 100 ml or 1 ml FCS-free 199 or DMEM medium per well.…”

Expression regulation of zebrafish interferon regulatory factor 9 by promoter analysis

“…The purified fusion protein was applied to immunize rabbits to raise a polyclonal anti-IRF9 antiserum according to previous reports (Jiang et al, 2009;Yu et al, 2010). Western blots were performed according to previous reports (Jiang et al, 2009;Liu et al, 2011;Sun et al, 2010Sun et al, , 2011. Briefly, protein samples were separated on 12% SDS-PAGE gels and then electrophoretically transferred to a PVDF membrane (Millipore).…”