Chicken fibroblast DF-1, macrophage-like HD11, and B-cell lymphoma DT40 cell lines were obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, United States) or RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) at 37°C and 5% CO₂. Chicken embryo fibroblasts (CEFs) were prepared from 9- to 11-day-old specific pathogen-free (SPF) chicken embryonated eggs (MERIAL, Beijing, China), as previously described (Qiu et al., 2011).

Newcastle Disease Virus strains LaSota/46 and Mukteswar were obtained from the China Institute of Veterinary Drug Control (Beijing, China). AIV strain H9N2 (A/Chicken/Shanghai/010/2008) was isolated from ducks in Shanghai in 2008 (Cheng et al., 2015a). The 50% tissue culture infective dose (TCID₅₀) for viruses were titrated onto DF-1 or MDCK cells using the Reed and Muench method. Virus titers were calculated by determining the dilution that yielded 50% of cells displaying cytopathic effects (Pizzi, 1950).

The chCMPK2 amplification primers were designed according to the National Center for Biotechnology Information (NCBI) reference sequence (GenBank: XM_015284945.3). The chCMPK2 cDNA was synthesized from total cellular RNA isolated from DF-1 cells. Then the entire ORF of the chCMPK2 gene was amplified using the primers chCMPK2_F and chCMPK2_R (Supplementary Table S1, synthesized in Sangon Biotech Co.). The PCR conditions used were as follows: an initial denaturation step at 95°C for 3 min, followed by 30 cycles of 95°C for 15 s, 66°C for 30 s, and 72°C for 1 min and one final extension step at 72°C for 5 min. PCR products were purified and inserted into the pMD19T vector for Sanger sequencing.

The PCR products were inserted into the EcoRI/BamHI sites of the p3xFLAG-CMV™-14 (pCMV-Flag) vector to generate the pCMV-Flag-CMPK2 expression plasmid. Successful insertion was confirmed by sequencing. To verify the expression of the recombinant plasmid, HEK293T cells were transfected with pCMV-Flag-CMPK2 or p3xFLAG-CMV™-14 empty vector by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) in accordance with the manufacturer’s instructions when the cell monolayer reached 80–90% confluence. At 24 h post-transfection, the cells were collected for Western blot (WB) assay. The FLAG-labeled chCMPK2 protein was detected using an anti-FLAG antibody (Cell Signaling Technology, Danvers, MA, United States).

According to the sequencing results of chCMPK2 and CMPK2 sequences of other animals obtained from NCBI, the nucleotide sequences were aligned with CLUSTALW and then a phylogenetic tree was constructed based on the amino acid sequences according to the maximum likelihood algorithm by MEGA7.

The amino acids of these sequences were uploaded to the EMBL-EBI website¹ for similarity analysis by using the multiple sequence alignment tools. The predicted human CMPK2 structure was obtained from the AlphaFold Protein Structure Database,² based on which the 3D structure of chCMPK2 was constructed on the Swiss Model website.³ All structural annotations were generated using PyMOL (version 1.74, Schrödinger).

Data of gene expression microarrays (GSE40100, GSE65231) were obtained from the GEO database. Differentially expressed genes (DEGs) were obtained by GEO2R using the cut-off standards |log₂(fold change)| > 1, p < 0.05.

Heat maps plot was drawn using the “pheatmap” packages in R version 4.0.3. Venn analysis was performed using the jvenn website.⁴

To test the chCMPK2 protein, mouse polyclonal serum against chCMPK2 was produced by the following steps. A highly conserved amino acid region (139–253) of chCMPK2 was chosen by aligning with the human and mouse CMPK2 amino acid sequences. This region was then cloned into pET28a (+) using EcoRI/SalI sites to generate pET28a-chCMPK2. The recombinant chCMPK2 protein, which was labeled with a His tag, was expressed in Escherichia coli BL21 cells and column-purified as described in the pET system manual. Polyclonal antiserum against chCMPK2 was raised in 6-week-old BALB/c mice, as described previously (Qiu et al., 2016). To minimize mouse suffering and distress, all invasive manipulations were carried out under anesthesia using 1% sodium pentobarbital at a dose of 50 mg/kg body weight. No unexpected deaths occurred during this study. The mice were euthanized by CO₂ inhalation for 5 min at the end of the study.

To measure the dynamic expression pattern of chCMPK2 after virus infection in different cells, cells seeded in 12-well plates were infected with AIV H9N2 or NDV strain Mukteswar and LaSota strain. At 6 and 12 h post-infection (hpi), cells were harvested, stored in TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and then subjected to RNA extraction according to the manufacturer’s instructions. Total cDNA was synthetized after RNA pellets were re-suspended in 50 μl RNase-free water and reverse transcribed with Oligo (dT) 18 Primer (Takara, Dalian, China) using Mo-MLV reverse transcriptase (Promega, Madison, WI, United States).

The cDNA of chicken tissues was collected in a previous study (Shi et al., 2020). SPF chickens were hatched from SPF eggs (MERIAL, Beijing, China) and randomly divided into uninfected and NDV-infected groups. At the age of 4 weeks, each chicken in the NDV-infected or H9N2-infected group was intramuscularly challenged with 0.2 ml containing 1 × 10⁵ EID₅₀ NDV strain LaSota/46 or AIV H9N2. The uninfected control group was mock-infected with PBS. At 3 days post-infection (dpi), the organs from five chickens were collected, including the spleen, bursa, thymus, kidney, liver, lung, duodenum, intestine, brain, and heart.

The qRT-PCR analysis was performed to determine the expression levels of chCMPK2 mRNA and viral RNA. The primers used for qRT-PCR are shown in Supplementary Table S2. CMPK2 levels and the levels of RNA encoding H9N2 NP were measured at different hpi, and CMPK2 mRNA levels were normalized to β-actin. The fold changes of relative mRNA levels were determined using the comparative 2^−ΔΔCt method as previously described (Schmittgen and Livak, 2008).

To determine the expression levels of chCMPK2 and viral NP proteins (TransGen Biotech, China), cells were washed thoroughly in PBS twice and then lysed in 2× SDS loading buffer. Lysates were resolved by 10% SDS-PAGE, after which the proteins were transferred to nitrocellulose membranes (Whatman International, Ltd.), which were blocked for 2 h with 2% BSA in Tris-buffered saline with Tween 20 (TBST) at room temperature. Then the membranes were incubated with rabbit anti-β-actin, mouse anti-chCMPK2, and anti-NP, followed by incubation with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, United Kingdom) using a Kodak imager (Carestream Health, Inc., Rochester, NY, United States).

chCMPK2-specific short interfering RNA (siRNA) and negative control (siNC) were designed and synthesized by Gene Pharma, Shanghai. The sequences of siCMPK2-1, siCMPK2-2, and siCMPK2-3 are shown in Supplementary Table S3. DF-1 cells were seeded into a 12-well plate and incubated for 12 h before transfection. Each well was washed twice with PBS and transfected with either 40 pmol siRNA mix or 40 pmol siNC with Lipofectamine 2000. At 36 h post-transfection, the cells were infected with AIV H9N2 at a multiplicity of infection (MOI) of 0.1. At 12 hpi, the cells were harvested to quantify the knockdown efficiency of chCMPK2 by qRT-PCR and WB assays.

DF-1 cells were seeded into 6-well glass bottom plates (Corning Incorporated, Corning, NY) the day before transfection. For mitochondrial colocalization analysis, cells were incubated with Mito-Tracker Red CMXRos (Beyotime Institute of Biotechnology, Jiangsu, China) for 30 min after transfection with pCMV-Flag-CMPK2 and pCMV-Flag for 36 h. After fixation with 4% formaldehyde, cells were permeabilized with 0.2% Triton X-100 and blocked in PBS with 3% BSA. Anti-Flag antibodies were used at a dilution of 1:500. Fluorophore-conjugated secondary antibodies and DAPI (4′,6-diamidino-2-phenylindole) were diluted 1:1,000 in PBS. Confocal laser scanning microscopy was performed with a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss Microimaging GmbH, Germany) fitted with a Plan Apochromat 63×/1.4 oil objective. Images were analyzed with Fiji software⁵ and ImageJ software. The experiments were repeated at least three times.

All experiments were performed in triplicate, and statistical analysis was conducted with GraphPad Prism 8 (GraphPad Software, San Diego, CA, United States). Data were analyzed with one-way analysis of variance (ANOVA) with Kruskal-Wallis comparison or two-way ANOVA with Tukey’s comparison. Statistical significance was defined as p < 0.05.

To identify undiscovered molecules involved in host innate immunity, we analyzed our previous transcriptomics data of NDV-infected DF-1 cells (Liu et al., 2018), and found that chicken CMPK2 was notably upregulated as one of the top five upregulated host genes, including OASL, APOLD1, IFIT5, Mx1, indicating that the IFN response was apparently activated, in response to NDV strain Herts/33 (Figure 1A). We then analyzed two public data in GEO database: GSE65231 (gene expression profiling data of the lung of H5N1-infected chicken; Ranaware et al., 2016) and GSE40100 (gene expression profiling data of the spleen under NDV infection; Hu et al., 2015) and identified 79 overlapping upregulated genes (4,955 in GSE65231 H5N1 vs. Mock, 446 in GSE40100 Herts/33 vs. Mock, and 664 in GSE40100 JS5/05 vs. Mock) which is showed in Venn plot (Figure 1B). In these common upregulated genes, CMPK2 ranks in the forefront with log₂ (fold-change) values of 5.45, 4.336017, and 5.2694117. The genes related to IFN and ISGs, including CMPK2 and several classic antiviral factors Mx1, TRIM25, and RASD2, among the top 20 are shown in a heat map (Figure 1C). Based on these data, we speculated that chicken CMPK2 may be involved in the immune response upon NDV and AIV infection.

The cDNA from AIV H9N2-infected DF-1 cells was used as a template to amplify the chCMPK2 gene. Our results showed that the chCMPK2 ORF consisted of 762 base pairs encoding 254 amino acids (Supplementary Figures S1A,B), which was the same as the reference sequence obtained from the chicken tissues (GenBank, accession No. XM_015284945.3). The molecular weight of chCMPK2 was around 33 kDa which was different from the predicted size of 28.03 kDa (Supplementary Figure S1C).

Alignment and phylogenetic analysis of chCMPK2 with other CMPK2 amino acid from different species indicated that chCMPK2 was highly conserved among birds, such as Centrocercus urophasianus, Lagopus leucura, Falco naumanni, and Corvus kubaryi. CMPK2 from Homo sapiens and Mus musculus form a subclade and were distant from chicken CMPK2, which is obviously distant from reptile sequences (Figure 2A). According to the full-length amino acid sequence alignment between birds and other species, the amino acid homology between chicken CMPK2 and other species was 34.43–69.57% (Table 1). Further multiple alignment showed that chicken CMPK2 shared 70.74, 68.62, and 72.08% amino acid identity to H. sapiens, M. musculus, and Panthera leo in the TMK domain, also known as thymidine monophosphate kinase (TMPK), whose functional domain consists of a P-loop, a catalytic site, and a lid motif (Table 2; Chen et al., 2008; Xu et al., 2008). According to the sequence of human CMPK2, the P-loop, the putative catalytic site, and the lid domain are located at amino acids 63–72, 137–149, and 167–180 in chicken CMPK2 (Figure 2B). Multiple sequence alignment revealed that the three active domains of chCMPK2 TMK shared 81.25–100% amino acid identity with those of other species (Table 3), which may indicate that this domain is highly conserved in different species and plays an important role on the function of chCMPK2.

A standard curve was generated using a tenfold serial dilution of the pCMV-Flag-CMPK2 plasmid, and the linear regression equation was Y = −5.251X + 51.495, with an R² value of 0.998, indicating a strong linear correlation. As shown in Figure 3A, chCMPK2 mRNA was highly expressed in the chicken macrophage line HD11; in contrast, lower expression of chCMPK2 mRNA was observed in other chicken cells, including DF-1 cells, CEFs, and B lymphocyte line DT40. It suggested that chCMPK2 mRNA was expressed in macrophages, which is consistent with the high expression of CMPK2 in human-derived macrophages cell line (THP-1; El-Diwany et al., 2018).

To obtain the expression profile of chCMPK2 in 4-week-old SPF chickens, the abundance of CMPK2 in organs was determined by normalizing the data with brain via using a comparative quantification method. The chCMPK2 mRNA was detected in most tissues examined and showed tissue-specific differences. Expression levels were highest in the duodenum (p < 0.001), followed by the liver and the immune organs lung, intestine, and kidney, whereas relatively low expression was detected in the brain, spleen, and bursa (Figure 3B). Our results revealed that chCMPK2 was highly expressed in the digestive system of chicken.

To determine whether chCMPK2 could be expressed in protein level, mouse polyclonal antiserum against chCMPK2 was generated by immunizing mice with recombinant pET28a-chCMPK2 protein expressed and purified from E. coli. To test the validity and specificity of this serum, DF-1 cells transfected with pCMV-Flag-CMPK2 were used for WB assays at 24 h post-transfection. Anti-chCMPK2 serum specifically recognized Flag-tagged chicken CMPK2, which displayed similar-sized target bands to the anti-Flag antibodies (Supplementary Figures S1C,D).

Previous studies have shown that CMPK2 was upregulated after stimulation with LPS, poly(I:C), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and SVCV (Liu et al., 2019). The expression of chCMPK2 in response to chicken virus infection were determined in this study. The qRT-PCR assay results confirmed that the AIV H9N2 infection induced a substantial fold upregulation of the mRNA of chCMPK2 at 6 and 12 hpi followed by the decrease at 18 and 24 hpi; while the expression of chCMPK2 protein increased from 6 to 24 hpi (Figure 4A). After NDV infection, the mRNA of chCMPK2 increased from 6 to 18 hpi in the group of Mukteswar infection and from 6 to 24 hpi in the group of LaSota infection and the chCMPK2 protein levels showed similar trends (Figure 4B). Further, we evaluated chCMPK2 expression in AIV and NDV-infected CEF and HD11 cells. The results showed that chCMPK2 mRNA levels were significantly upregulated in CEF and HD11 cells during AIV H9N2 infection, while no significant increase was detected in NDV-infected HD11 cells (Figures 4E,F). The significant upregulated levels of mRNA and protein were obtained after stimulation with the double stranded RNA (dsRNA) analog poly(I:C; Figure 4G).

To detect the expression of chCMPK2 in vivo during virus infection, the cDNAs samples of tissues, including brain, spleen, bursa, kidney, duodenum, lung, heart, and thymus were collected from chickens infected with AIV H9N2 or NDV LaSota at 3dpi in our previous studies, which was kept in −80°C (Shi et al., 2020). SPF chickens were infected by AIV H9N2 and NDV LaSota in a dose of 1 × 10⁶ EID₅₀ and 1 × 10⁵ EID₅₀, respectively, by intranasal inoculation. The high expression level of CMPK2 in chicken intestine and the upregulation in immune-related tissues including thymus, spleen and lung after intranasal inoculation of AIV H9N2 were demonstrated by qRT-PCR (Figure 5A). In addition to the increased expression of CMPK2 in immune organs, such as kidney, intestine and thymus, CMPK2 levels still remained at a high level in the heart, bursa and duodenum after NDV LaSota infection (Figure 5B). Collectively, these findings suggest that chCMPK2 is involved in the host immune response against virus infection.

According to previous studies, the upregulation of CMPK2 mRNA can be induced by poly(I:C), LPS, and IFN-β in human and fish cells (Collins et al., 2007; El-Diwany et al., 2018; Kim et al., 2021). In the present study, we found that the CMPK2 mRNA levels increased after poly(I:C) stimulation as well as upon virus infection, which was consistent with the increase of IFN-β levels (Figures 4C,D,H). To determine whether chCMPK2 expression could be regulated in response to IFN-β or IL stimulation, the CMPK2 mRNA levels in DF-1 cells were detected after stimulation with plasmids expressing chicken IFN-α, IFN-β, IFN-γ, IL-6, IL-8, and IL-1β. Compared to the results in mammals and fish, the chCMPK2 mRNA level is a strong indicator of type I IFN and type III IFN levels, but shows a minimal response to IL stimulation (Figure 6A). To determine whether the expression of chCMPK2 was regulated by MDA5 and IFN-β, DF-1 cells were stimulated with specific siRNA targeting MDA5 or IFN-β for 24 h and then treated with poly(I:C). The effect of siRNAs was tested by qRT-PCR (Supplementary Figure S2). Both mRNA and protein-expression levels of chCMPK2 were markedly reduced after siRNA-mediated knockdown of MDA5 or IFN-β and poly(I:C) treatment (Figure 6B). All these data indicated that chicken CMPK2 was associated with MDA5 and IFN-β expression. Consistent with previous findings in human, these data confirmed that chCMPK2 was an MDA5/IFN-β-inducible gene in chicken cells.

In SVCV-infected FHM cells, overexpression of CMPK2 has significant antiviral effects. The opposite effects were observed when CMPK2 was knocked-down (Liu et al., 2019). To further explore the role of chCMPK2 in antiviral immune responses, pCMV-Flag-CMPK2 or pCMV-Flag (control) was transfected into DF-1 cells, and at 24 h post-transfection the cells were infected with AIV H9N2 at an MOI of 0.1. Then qRT-PCR and WB analysis were performed and the results showed that overexpression of CMPK2 significantly suppressed AIV H9N2 replication compared with the Flag control at both mRNA and protein levels at 6, 12, and 24 hpi (Figures 7A,B). Furthermore, it is noted that the antiviral effect of chCMPK2 appeared to be in a dose-dependent way (Figures 7C,D).

Further, chCMPK2 gene was knocked down in DF-1 cells by transfection with CMPK2-specific siRNA, the knockdown efficiency of gene transcription was approximately 60%, and the expression level was approximately 70% lower (Supplementary Figure S2C). To obtain more functional evidence for chCMPK2 in antiviral responses, DF1 cells were treated with chCMPK2 siRNA followed by transfection with AIV H9N2. As shown in Figures 7E,F, viral NP protein increased in chCMPK2 knockdown cells compared to control cells in 6 and 12 hpi, and which were consistent with the results of qRT-PCR. Together, these results demonstrated that chCMPK2 was required for host defense against AIV H9N2 infection in vitro.

In order to determine the antiviral effect of chCMPK2 on NDV, DF-1 cells were infected with LaSota and Mukteswar at an MOI of 0.1 after transfection with chCMPK2 for 24 h. There was no significant change in the mRNA levels of NDV NP (Figures 7G,H) at 6 and 12 dpi. However, the viral NP protein levels of LaSota and Mukteswar were significantly reduced at both 6 and 12 dpi. These data indicated that chCMPK2 may be also an antiviral molecule against NDV.

To determine the subcellular localization of CMPK2, pCMV-Flag-CMPK2 was transfected into DF-1 cells. The high expression of CMPK2 in transfected DF-1 cells was verified by WB (Supplementary Figure S1D), and the fluorescence was observed under a confocal microscope. As shown in Figure 8A, the green fluorescence of the CMPK2 fusion protein was distributed in the cytoplasm and nucleus of DF-1 cells. In previous reports, human CMPK2 and fish CMPK2 was localized in the mitochondria of HeLa cells and FHM cells (Xu et al., 2008; Liu et al., 2019). In the present study, mitochondria were stained by Mito Tracker, and chicken CMPK2 was partially localized in the mitochondria of DF-1 cells.

To determine the key domain of chCMPK2 for its antiviral effect against AIV H9N2, full-length chCMPK2 was truncated in four ways (Figure 8B). DF-1 cells were transfected with four truncated chCMPK2 mutant plasmids to identify the antiviral effects and the changes in subcellular localization were observed. As shown in Figure 8C, there were no influences on the subcellular localization of chCMPK2 after the C-terminal 159 amino acids were truncated (CMPK2-Δ96-161aa and CMPK2-Δ137-253aa), while no nuclear localization was observed when the N-terminal region was truncated (CMPK2-Δ1-30aa and CMPK2-Δ35-95aa). It is inferred that the nuclear localization signal sequence was located in the first 95 amino acids on the N-terminus of chCMPK2. However, the nuclear localization of chCMPK2 was not related to its antiviral effect, because CMPK2-Δ1-30aa and CMPK2-Δ35-95aa showed similar antiviral activity against AIV with that of full-length chCMPK2. As shown in Figure 8E, the antiviral effects of CMPK2-Δ96-161aa and CMPK2-Δ137-253aa were significantly inhibited, suggesting the fragments determining the antiviral function of chCMPK2 were located at the C-terminus of chCMPK2, which consisted of an evolutionarily conserved TMK domain.

To identify the key amino acid for the function of TMK domain, the 3D models of the chCMPK2 and huCMPK2 TMK domains were constructed using homology modeling on the Swiss-model website. The positions of the three catalytic regions (P-loop, catalytic site, and lid domain) are consistent with those of humans (Figure 8D). The alignments revealed that the conserved putative catalytic site motif of chCMPK2 contained the highly conserved Asp135, Arg136, Trp138, His139, Ser140, Thr141, and Ala142 residues (Figure 2B). Previous studies of mammalian CMPK2 indicated that the highly conserved aspartate (D) residue in its catalytic pocket, which is located in the putative catalytic site, could determine the catalytic activity of huCMPK2 (Zhong et al., 2018). The catalytic activity and the activation of NLRP3, which was induced by LPS-stimulated mtDNA replication, could be inhibited by replacing the highly conserved aspartate (D) residue with alanine (A) (Zhong et al., 2018). According to the amino acid sequence alignment results above (Figure 2B; Table 3), chCMPK2 shows the same highly conserved aspartate residue at the 135th site, and chCMPK2 3D models show that chCMPK2 D135 residues are located in a pocket which coincides with the position of the huCMPK2 D330 residue (Figure 8D). To further investigate the role of the Asp135 site in the antiviral function of chCMPK2, a mutant plasmid named CMPK2 (D135A), in which Asp135 was replaced with alanine (A), was generated and transfected into DF-1 cells. The mutant showed significantly weaker effects on AIV H9N2 replication than wild-type chCMPK2 in mRNA and protein expression levels (Figure 8F). Taken together, our results indicated that the antiviral function of chCMPK2 was dependent on the catalytic activity of the TMK domain.