Host protein PRPS2 interact with the non-structural protein p17 of Avian Reovirus and promote viral replication

Abstract

Avian reovirus (ARV) is highly prevalent in healthy poultry flocks and has been linked to viral arthritis/tendonitis, dwarf syndrome, chronic respiratory disease, and immunosuppression in avian species, resulting in significant economic losses within the poultry industry. The non-structural protein p17 encoded by ARV induces cellular autophagy and facilitates viral proliferation, playing a pivotal role in viral pathogenesis. To further elucidate the pathogenic mechanism basis of ARV p17 protein function, we employed a yeast two-hybrid system to identify Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) as an interacting host protein with p17. In this study, we validated the interaction between PRPS2 and p17 using laser confocal microscopy, coimmunoprecipitation, and GST-Pulldown assays. Moreover, our findings demonstrate that the C-terminal region of PRPS2 is responsible for its binding to the p17 protein. Intriguingly, ARV infection significantly upregulated PRPS2 expression levels. Additionally, PRPS2 was shown to have a substantial impact on ARV replication; overexpression of PRPS2 increased ARV replication while knockdown of PRPS2 resulted in decreased quantities of ARV particles. Furthermore, our findings suggest that this process involves cellular apoptosis as a potential mechanism underlying these observations. Overall, this research provides valuable insights into elucidating the function of the p17 protein and sheds light on the pathogenic mechanism involving ARV-induced cellular apoptosis while offering novel perspectives for exploring therapeutic targets against ARV.

Keywords: Avian reovirus; PRPS2; Replication; p17 protein.

Fig. 1

ARV p17 interacts with PRPS2. (A) The blue colonies of yeast strains indicate the positive proteins screen by yeast two-hybrid assay. (B) 293T cells were co-transfected with PRPS2-Flag and p17-Myc plasmids for 48 h and harvested. Cell lysates were immunoprecipitated with Flag or p17 antibody, then detect target proteins through Western blotting analysis. (C) GST pulldown assay. Glutathione beads conjugated to GST or the GST-p17 protein fusion protein were incubated with recombinant PRPS2-Flag protein. After washing, proteins were eluted from the beads, and protein PRPS2-Flag was detected by Western blotting with an anti-Flag mAb. (D) Colocalization of Flag-PRPS2 and ARV p17 protein. 293T cells were cotransfected with Flag-PRPS2 and p17-Myc for 24 h, then incubated with anti-Flag and anti-Myc primary antibodies and immunostained with Cy3- or FITC-labeled secondary antibodies. Finally, the cells were analyzed by confocal microscopy.

Fig. 2

The C-Terminus of the ARV p17 Protein mediate the interaction with PRPS2. (A) Schematic representation of the protein domains of the ARV p17 protein. (B) 293T cells were cotransfected with PRPS2-Flag and different part of p17-Myc plasmids for 48 h and harvested. Cell lysates were immunoprecipitated with Myc antibody, then detect target proteins by Western blotting analysis. (C) 293T cells were transfected with different parts of the p17-Myc vector and studied by electron microscopy. Yellow arrows indicate the structures with the characteristics of autophagosomes.

Fig. 3

Overexpression PRPS2 enhances ARV replication. (A) Vero cells were infected with ARV at a MOI of 1 for different times, and the intracellular expression level of PRPS2 was quantified by qRT-PCR. (B) Vero cells were infected with ARV at a MOI of 1 for different times, and the quantity of ARV replication was assayed by qRT-PCR. (C) Intracellular PRPS2 expression after transfection with the pcDNA3.1-PRPS2 plasmid in Vero cells was revealed by qRT-PCR. (D) Following transfection with pcDNA3.1-PRPS2 for different durations, Vero cells were infected with ARV at indicated time points, and the quantity of ARV replication was measured by qRT-PCR. (E) The ARV p17 protein levels in Vero cells were analyzed by Western blotting. The numbers represent the ratio of p17/GAPDH in different ARV infection time.The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey's HSD post hoc analysis. ***P < 0.001, compared with the mock group.

Fig. 4

Knockdown PRPS2 expression decreases ARV replication. (A) Intracellular PRPS2 expression after transfection with si-PRPS2 or siRNA-NC at different time in Vero cells was revealed by qRT-PCR. (B) After transfected with si-PRPS2 24 h, the Vero cells infeciton ARV at indicated time. The quantity of ARV replication in Vero cells was revealed by qRT-PCR. (C) The ARV p17 protein level in PRPS2-knockdown Vero cells was analyzed by Western blotting. The numbers represent the ratio of p17/GAPDH in different ARV infection time. Results are presented as the means ± SD of data from three independent experiments. (***P < 0.001).

Fig. 5

PRPS2 protein promote ARV infection induce apoptosis in Vero cells. (A) The apoptotic of Vero cells were determined by AnnexinV-FITC and PI double staining assay. (B) Percentage of apoptotic rate of cells measured by flow cytometry. Results are presented as the means ± SD of data from three independent experiments. (***P < 0.001).