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Dev Comp Immunol
2023 Apr 01;141:104648. doi: 10.1016/j.dci.2023.104648.
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Identification and functional characterization of protein kinase R (PKR) in amphibian Xenopus tropicalis.
Gan Z
,
Xu X
,
Tang S
,
Wen Q
,
Jin Y
,
Lu Y
.
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As one of interferon-induced serine/threonine kinases, the protein kinase R (PKR) plays vital roles in antiviral defense, and functional features of PKR remain largely unknown in amphibians, which suffer from ranaviral diseases in the last few decades. In this study, a PKR gene named Xt-PKR was characterized in the Western clawed frog (Xenopus tropicalis). Xt-PKR gene was widely expressed in different organs/tissues, and was rapidly induced by poly(I:C) in spleen, kidney, and liver. Intriguingly, Xt-PKR could be up-rugulated by the treatment of type I and type III interferons, and the transcript level of Xt-PKR induced by type I interferon was much higher than that of type III interferon. Moreover, overexpression of Xt-PKR can suppress the protein synthesis and ranavirus replication in vitro, and the residue lysine required for the translation inhibition activity in mammalian PKR is conserved in Xt-PKR. The present study represents the first characterization on the functions of amphibian PKR, and reveals considerable functional conservation of PKR in early tetrapods.
Fig. 1. Multiple alignments of PKR protein sequence from X. tropicalis. Identical amino acids and amino acids with high and low similarity are illustrated with “*”, “:”, and “.”, respectively. Tandem dsRBMs and conserved catalytic subdomains are highlighted in solid lines above the alignment, and conserved residues that are crucial for kinase activity and a conserved LFIQMEWCE motif are indicated in black and grey shadow, respectively. The amino acid sequences from different species are named accordingly and abbreviated as: zebrafish (D. rerio, as Dr), Western clawed frog (X. tropicalis, as Xt), chicken (G. gallus, as Gg), mouse (M. musculus, as Mm), and human (H. sapiens, as Hs).
Fig. 2. The expression of PKR gene in different organs/tissues of healthy clawed frogs (A), frogs treated with poly(I:C) (B), and frogs treated with type I or type III IFNs (C) (n = 3). Gene expression was measured by qRT-PCR, and the expression data were normalized against the level of β-actin. Injection of APBS (B) or empty vector-expression protein (C) served as control group, and fold changes were calculated relative to control group. Data were expressed as mean ± SEM, with * indicating p < 0.05.
Fig. 3. Translation inhibition activity of PKR from X. tropicalis. HEK293T cells were separately transfected with pcDNA3.1 vector subcloned with the entire ORF of Xt-PKR (Xt-PKR-WT), the C-terminal catalytic kinase domain deletion mutant (Xt-PKR-ΔC), the N-terminal dsRBD deletion mutant (Xt-PKR-ΔN), and the catalytically inactivated mutant with replacement of lysine (K) by arginine (R) (Xt-PKR-ΔN-K305R), or empty pcDNA3.1, together with luciferase plasmid pGL3 promoter and pRL-TK. Luciferase activity of transfected cells was determined by using the luciferase detection kit. Data were normalized to the Renilla luciferase activity and expressed as mean ± SEM, with * indicating p < 0.05.
Fig. 4. Antiviral effect of PKR from X. tropicalis against FV3 in A6 cells. (A) Xt-PKR-overexpressing A6 cells were infected with FV3, and the culture supernatants were removed and the cell monolayers were fixed with paraformaldehyde and stained with crystal violet. (B) A6 cells were separately transfected with pcDNA3.1 vector subcloned with the entire ORF of Xt-PKR (Xt-PKR-WT), the C-terminal catalytic kinase domain deletion mutant (Xt-PKR-ΔC), the N-terminal dsRBD deletion mutant (Xt-PKR-ΔN), and the catalytically inactivated mutant with replacement of lysine (K) by arginine (R) (Xt-PKR-ΔN-K305R), or empty pcDNA3.1, and were infected with FV3. Then cells were subjected to three rounds of sequential freeze-thaw lysis, and the resulting homogenates were collected for the determination of virus titer by standard plaque assays. The data are representative of three independent experiments and are presented as mean ± SEM, with * indicating p < 0.05.
