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. 2011 Dec;85(23):12769-80.
doi: 10.1128/JVI.05849-11. Epub 2011 Sep 21.

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response

Ting-Kai Liu  1 , Yi-Bing Zhang, Ying Liu, Fan Sun, Jian-Fang Gui
Affiliations

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response

Ting-Kai Liu et al. J Virol. 2011 Dec.

Abstract

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). 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 eIF2α 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 eIF2α. 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 eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α 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.

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Figures

Fig. 1.
Fig. 1.
Coexistence of fish PKR and PKZ. (A) Genomic coexistence of tandemly arranged PKR and PKZ genes in Danio rerio and Carassius auratus. The arrows indicate the 5′ to 3′ orientation of the genes. The approximate sizes of the intergenic regions are indicated below the maps. (B) Taxonomic tree of species used in the phylogenetic analysis. The taxonomic tree is constructed by NCBI taxonomy (http://www.ncbi.nlm.nih.gov/taxonomy). Arrows indicate the species in which PKZ genes have been found. (C) Phylogenetic relationship of PKR and PKZ proteins. The phylogenetic tree was constructed by maximum likelihood method from the multiple-sequence alignment of the kinase domains of PKR and PKZ without the kinase insert using the MUSCLE approach. Human and zebrafish PERK were used as outgroups for rooting the phylogenetic tree. The abbreviations used here are shown in parentheses immediately after the species name in panel B. The accession numbers of the sequences used in panel C are given in Materials and Methods.
Fig. 2.
Fig. 2.
Induction of fish PKR and PKZ by poly(I · C) and IFN. (A) Schematic illustration of promoter-driven luciferase (Luc) constructs PKRpro-luc (PKRp-luc) and PKZpro-luc (PKZp-luc). (B and C) Activation of the PKR and PKZ promoters by poly(I · C) (polyI:C) or rIFN. CAB cells in each well of 24-well plates were cotransfected with 0.05 μg pRL-TK and 0.5 μg PKRpro-luc or PKZpro-luc in 500 μl medium. At 24 h posttransfection (hpt), the cells were stimulated by transfection of poly(I · C) (0.4 μg/ml) (B) or the addition of rIFN (20 ng/ml) (C). At 48 hpt, the cells were harvested for detection of luciferase activity. Rel. lucif. Act., relative luciferase activity. (D to F) Induction of fish PKR and PKZ proteins by poly(I · C) or rIFN. CAB cells seeded in 6 well-plate were mock transfected with 1.6 μl PBS (D) or transfected with 1.6 μl of a 1-μg/μl poly(I · C) solution (E) or 20 ng rIFN was added to 1 ml medium (F). The cells were harvested at 0, 24, 48, or 72 hpt for detection of PKR and PKZ by Western blot analysis. (G) Requirement of ongoing IFN synthesis for transcriptional induction of fish PKR and PKZ genes. CAB cells seeded in 6-well plates were mock transfected with 1.6 μl PBS or transfected with 1.6 μl of a 1-μg/μl poly(I · C) solution in 1 ml medium or treated with rIFN at 20 ng/ml in the absence or presence of 8 μg/ml cycloheximide (CHX) for 24 h, and total RNA was extracted. Reverse transcription-PCR (RT-PCR) was employed to detect PKR and PKZ expression. (H) Involvement of the Stat1 pathway in PKR and PKZ induction. CAB cells stably transfected with pcDNA3.1 or Stat1-ΔC were treated with 5 ng/ml of rIFN for 10 h and then harvested for detection of PKR and PKZ protein by Western blot analysis.
Fig. 3.
Fig. 3.
Concomitant induction of eIF2α phosphorylation by poly(I · C) and IFN. (A to C) Concomitant induction of eIF2α phosphorylation by poly(I · C) and rIFN. Samples loaded here are the same as those in Fig. 2D to F, respectively, but phosphorylated eIF2α (p-eIF2α) and total eIF2α were detected by Western blot analysis. (D) Involvement of the Stat1 pathway in eIF2α phosphorylation by poly(I · C) and rIFN. The same samples shown in Fig. 2H were loaded here for Western blot analysis of total eIF2α or phosphorylated eIF2α. (E) Direct interaction of eIF2α with PKR or PKZ. CAB cells seeded in 10-cm dishes were mock induced by transfection of 8 μl PBS [poly(I · C) absent] or induced by transfection of 8 μl × 1 μg/μl poly(I · C) [poly(I · C) present] in 5 ml medium for 48 h and then harvested for coimmunoprecipitation assay by anti-eIF2α antibody. PKR, PKZ, p-eIF2α, and eIF2α were analyzed by Western blotting in both immunoprecipitation (IP) samples (left blots) and input samples (right blots). (F to H) CAB cells were transfected with poly(dG · dC) which mimics Z-DNA (F), poly(dA · dT) which mimics B-DNA (G), or unsheared calf genomic DNA (g-DNA) (H), harvested at 0, 24, 48, and 72 h posttransfection (hpt), and subjected to immunoblotting with antibodies specific to PKR, PKZ, actin, p-eIF2α, and eIF2α. The relative levels of eIF2α phosphorylation as determined by quantitative densitometry are shown below the blots. The value for the sample at 0 hpt is set at 1 as a reference with which other samples are compared.
Fig. 4.
Fig. 4.
Inhibition of reporter protein synthesis by fish PKR and PKZ. CAB, CO, EPC, COS-7, and 293T cells, seeded in 24-well plates, were cotransfected with the pGL3 plasmid (200 ng) and the expression vectors (200 ng) containing the corresponding inserts indicated by the illustration shown to the left of the bar graph. The K→R mutation that abolishes kinase activity but does not affect dimerization is indicated by a black asterisk. Luciferase activity was normalized by protein contents. A control experiment by using empty vector was set at 100%. Each bar shows the average ± standard error (error bar) for three independent experiments.
Fig. 5.
Fig. 5.
Inhibition of GCRV propagation by overexpression of fish PKR or PKZ. (A) CAB cells in each well of 6-well plates were cotransfected with a total amount of 1.6 μg of pcDNA3.1, PKR, PKZ, or PKR plus PKZ (1:1) plasmids in 1 ml fresh medium. At 0, 24, 48, and 72 hpt, cells were harvested for detection of PKR and PKZ proteins by Western blot analysis. (B) Phosphorylated eIF2α and total eIF2α were measured in the samples as indicated above for panel A. The relative levels of eIF2α phosphorylation as determined by quantitative densitometry are shown below the blots. The sample of 0 hpt is set at 1 as a reference with which other samples are compared. (C) The other group of transfected cells in 6-well plates was subjected to GCRV infection (each well with 1 ml of a solution with 104 TCID50/ml), and the supernatants were collected at 12, 24, 48, and 72 hpt for detection of virus titers by TCID50 assays.
Fig. 6.
Fig. 6.
Effects on GCRV replication by knockdown of PKR or PKZ. (A and B) CAB cells seeded in 24-well plates were delivered with control MO (mock), PKR-MO, PKZ-MO, or PKR-MO plus PKZ-MO (detailed in Materials and Methods) and induced by rIFN at 20 ng/ml for 12 h and subjected to immunoblot analysis. (C to E) The other group of MO-delivered cells were subjected to GCRV infection (each well with 1 ml of a solution with 200 TCID50/ml) for 48 h. While the cells were subjected to Western blot analysis (C and D), the supernatants were collected for detection of virus titers by TCID50 assays (E). The relative levels of eIF2α phosphorylation as determined by quantitative densitometry are shown below the blots in panels B and D.
Fig. 7.
Fig. 7.
Interaction of poly(I · C) with the N terminus of PKR but not with the N terminus of PKZ. The N terminus of PKR containing the three dsRBDs (PKR-N) and the N terminus of PKZ containing the two Z-DNA binding domains (ZBDs) (PKZ-N) and the C terminus of PKR containing only the kinase domain (PKR-C) were constructed and inserted into pcDNA3.1(+)-myc/His (Vector) at the EcoRI/KpnI sites. The primers are shown in Table S1 in the supplemental material. COS-7 cells seeded in 10-cm dishes were transfected with 8 μg each of the constructs as well as pcDNA3.1 by 20 μl Lipofectamine 2000. Cells were harvested using the same procedures as those used in the CoIP experiment. The poly(I · C) pulldown assay was carried out by the protocol given in our previous publication (44). Equal amounts of total protein were used in each pulldown assay. Lanes: inp, input (lysate before pulldown assay); pd, complexes that are pulled down by poly(I · C). IB, immunoblotting.
Fig. 8.
Fig. 8.
Independent association of fish PKZ and PKR. (A) PKR-HA, PKZ-HA, or vector (4 μg each) was cotransfected into 293T cells, seeded in 10-cm dishes with PKZ-myc or PKR-myc (4 μg each) in 5 ml medium, and the cells were stimulated by transfection of 0.2 μg/ml poly(I · C) at 8 hpt. The cells were harvested at 24 hpt and subjected to anti-HA IP assay. HA, myc, and human PKR (hPKR) antibodies were employed to analyze the input (top blots) and IP samples (bottom blots). αHA, anti-HA antibody. (B) 293T cells seeded in 10-cm dishes were transfected with 8 μg PKR-HA alone in 5 ml medium and stimulated by transfection of 0.2 μg/ml poly(I · C) at 8 hpt. The cells were harvested at 24 hpt and subjected to anti-HA IP or anti-IgG IP as a control. HA, PKR, eIF2α, and human PKR antibodies were employed in the Western blots. (C) Parallel experiment to the experiment shown in panel B but with PKZ-HA plasmid and PKZ antibody. Other conditions were the same as in panel B. N.S, nonspecific. (D) CAB cells seeded in 10-cm dishes were transfected with 8 μg PKZ-HA alone in 5 ml medium and stimulated by transfection of 0.2 μg/ml poly(I · C) at 8 hpt. The cells were harvested at 48 hpt and subjected to anti-HA IP or anti-IgG IP as a control. HA, PKZ, eIF2α, and crucian carp PKR antibodies were employed in Western blotting (immunoblotting [IB]).
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