An OTU deubiquitinating enzyme in Eimeria tenella interacts with Eimeria tenella virus RDRP

Abstract

Background: Chicken coccidiosis, a disease caused by seven species of Eimeria (Apicomplexa: Coccidia), inflicts severe economic losses on the poultry industry. Eimeria tenella is the one of the most virulent species pathogenic to chickens. Many parasitic protozoans are parasitised by double-stranded (ds) RNA viruses, and the influence of protozoan viruses on parasitic protozoans has been extensively reported. E. tenella RNA virus 1 (Etv) was identified in E. tenella, and the complete genome sequence of Etv was analysed. Here, we screened Etv-RNA-dependent RNA polymerase (RDRP)-interacting host protein E. tenella ovarian tumour (OTU) protein-like cysteine protease (Et-OTU) using a yeast two-hybrid system with pGBKT7-RDRP plasmid serving as bait. A previous study demonstrated that Et-OTU could regulate the telomerase activity of E. tenella, indicating that Et-OTU affects E. tenella proliferation. However, whether Etv-RDRP affects the molecular biological characteristics of E. tenella by interacting with OTU remains unclear.

Results: We obtained seven positive clones from the initial screen, and six of the seven preys were identified as false-positives. Finally, we identified an RDRP-associated protein predicted to be an E. tenella OTU protein. A α-galactosidase assay showed that the bait vector did not activate the GAL4 reporter gene, indicating no autoactivation activity from the RDRP bait fusion. Pull-down and co-immunoprecipitation assays verified the interaction between Et-OTU and Etv-RDRP both intracellularly and extracellularly. Additionally, Et-OTU was able to deconjugate K48- and K6-linked di-ubiquitin (di-Ub) chains in vitro but not K63-, K11-, K29-, or K33-linked di-Ub chains. The C239A and H351A mutations eliminated the deubiquitinase (DUB) activity of Et-OTU, whereas the D236A mutation did not. Additionally, when combined with RDRP, the DUB activity of Et-OTU towards K48- and K6-linked chains was significantly enhanced.

Conclusion: Etv-RDRP interacts with Et-OTU both intracellularly and extracellularly. Etv-RDRP enhances the hydrolysis of Et-OTU to K6- or K48-linked ubiquitin chains. This study lays the foundation for further research on the relationship between E. tenella and Etv.

Keywords: Deubiquitinase; Eimeria tenella; Enhanced; Et-OTU; Etv-RDRP; Interaction; Mutation.

Fig. 1

Construction of RDRP bait plasmids and testing of RDRP expression, toxicity, and autoactivation in yeast cells. a Agarose gel electrophoresis analysis of the bait plasmids pGBKT7-RDRP and pGBKT7. M: DNA marker; Lane 1: bait plasmid pGBKT7-RDRP digested with NdeI and SalI; Lane 2: pGBKT7 vector. b Immunoblot analysis of total proteins in the yeast strain Y187 transformed with the indicated plasmids. Lane 1: pGBKT7-RDRP; Lane 2: pGBKT7 vector. c Determination of the autoactivation and toxicity of RDRP bait in yeast cells. Yeast strain Y187 was transformed with pGBKT7-RDRP (upper) or pGBKT7 (lower) and grown on SD/-Trp and SD/-Trp/-X-α-gal plates. Lack of autoactivation is indicated by white colonies (or pink colonies) on SD/-Trp and SD/-Trp/-X-α-gal plates. Toxic bait is indicated by colonies significantly smaller than those containing the pGBKT7 empty vector

Fig. 2

Confirmation of both intracellular and extracellular interactions between RDRP and host proteins. a Yeast two-hybrid testing of RDRP-host protein interactions. Yeast strain Y187 was co-transformed with pGADT7-RDRP and pGBKT7-prey and plated on SD/-Trp/-Leu/-His/-ADE/-x-α-gal plates. Positive interactions were indicated by the presence of blue colonies. The co-transformation of pGBKT7-53 and pGADT7-T was used as a positive control, whereas the co-transformation of pGBKT7-Lam and pGADT7-T was used as a negative control. b GST pull-down assay confirming extracellular interactions between RDRP and OTU. The binding of His-RDRP to GST-OTU immobilised on glutathione beads was determined by Western blot analysis. The total protein eluted from the beads was probed with an anti-His antibody as indicated. c The intracellular interaction between RDRP and host OTU proteins as determined by coimmunoprecipitation. DH10Bac bacteria were transfected with pFast-HTA-RDRP and pFast-dual-OTU to construct the corresponding recombinant bacmids. The recombinant bacmids were then extracted and transfected into Sf9 cells. The recombinant baculoviruses were incubated with transfected cells at 27 °C and harvested every 7 days. Third-generation viruses were employed to infect Sf9 cells for protein expression. After co-transfection of OTU and RDRP recombinant baculoviruses for 48 h, cell lysates were analysed by Western blotting with anti-His and anti-GST antibodies. Immunoprecipitation was performed with an anti-His antibody (d) and an anti-GST antibody (e) and detected by Western blotting with anti-His and anti-GST antibodies. Untransfected cells were used as controls

Fig. 3

Conserved sequence alignment of catalytic Cys, Asp and His residues of the OTU family of DUBs. Amino acid alignment of TgOTUD3A (Toxoplasma gondii; GenBank: EPR62955.1), Otubain 2 (human; SW: Q96DC9), Otubain 1 (human; SW: Q96FW1), A20 (human; SW: P21580), Cezanne (human; SW: Q9NQ53) and VCIP135 (rat; SW: Q8CF97). The critical amino acid residues comprising the catalytic triad (Asp, Cys, and His) are highly conserved across these species (black asterisks) despite their evolutionary distance. The catalytic residues Asp, Cys, and His were mutated to generate Et-OTU (C239A), Et-OTU (D236A) and Et-OTU (H351A)

Fig. 4

Cleavage assays of purified Et-OTU on di-Ub WT substrates in vitro. a Purified Et-OTU DUB was incubated with the K48-, K63-, K11-, K29-, K33-, or K6-linked di-Ub chains for 30 min and resolved by Western blotting. b Purified Et-OTU DUB was incubated individually with the K48- and K6-linked di-Ub chains for the indicated times. The hydrolytic efficiency of Et-OTU DUB towards K6-linked di-Ub chains was higher than that of Et-OTU DUB towards K48-linked di-Ub chains. c Catalytic residues (Cys residue 239, Asp residue 236 and His residue 351) of Et-OTU were individually mutated to Ala. Purified OTUD236A, OTUC239A, OTUH351A and WT OTU DUBs were individually incubated with K48 and K6 di-Ub chains for 30 min

Fig. 5

Et-OTU DUB activity was enhanced by Etv-RDRP/Et-OTU complexes in vitro. In vitro deubiquitination assays were performed using Etv-RDRP in combination with Et-OTU and the Et-OTU protein purified from Sf9 as previously described. K48- and K6-linked di-Ub chains were individually used as substrates at a final concentration of 5 μM. The final concentrations of Et-OTU and Etv-RDRP in combination with Et-OTU in the reaction were both 5 μM. a Etv-RDRP in combination with Et-OTU and the Et-OTU protein was purified using glutathione-Sepharose 4B and individually analysed by Western blot. b In total, 10 μl of both purified Et-OTU DUB and purified RDRP/OTU complexes were separately incubated with 10 μl of K48-linked di-Ub chains for 30 min and resolved by Western blotting. The hydrolytic activity of Et-OTU DUB towards K48-linked di-Ub chains was analysed using ImageJ software. c In total, 10 μl of both purified Et-OTU DUB and purified RDRP/OTU complexes were separately incubated with 10 μl of K6-linked di-Ub chains for 30 min and resolved by Western blotting. The hydrolytic activity of Et-OTU DUB towards K6-linked di-Ub chains was analysed using ImageJ software