Interaction of porcine circovirus-like virus P1 capsid protein with host proteins

Abstract

Background: Porcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue.

Results: Five cellular proteins (EEP, Ral GDS, Bcl-2-L-12, CPS1, and one not identified) were found to interact with P1 Cap. The interaction between Cap and Ral GDS was confirmed by co-immunoprecipitation.

Conclusions: Our data are likely to support the future investigation of the underlying mechanism of P1 infection and pathogenesis.

Keywords: Cap; Porcine circovirus-like virus P1; cellular protein; co-immunoprecipitation; yeast two-hybrid assay.

Fig. 1

PCR products of the 24 randomly selected clones in 1.0 % agarose gel electrophoresis. DL12000 (Takara) was used as the marker. An enlarged marker is shown on the right side of the figure. The number of the lanes indicates the 24 randomly selected clones

Fig. 2

Detection of self-activation of the bait protein. The AH109 yeast cells that were cotransformed with pGADT7 and pGBKT7-ORF1, pGADT7-LargeT and pGBKT7-p53 (as a positive control), pGADT7-LargeT and pGBKT7-laminC (as a negative control), were plated on SD-TL and SD-TLHA medium for the auto activation test. Yeast co-transfected with plasmids pGADT7 and pGBKT7-ORF1 cannot grow on SD-TLHA medium and did not turn blue in the β-galactosidase assay, indicating that pGBKT7-ORF1 does not autonomously activate the reporter genes in yeast cells without a prey protein. The images were cropped, and full-length images are included in Supplementary Fig. 2a to 2c

Fig. 3

Yeast two-hybrid assay with P1 Cap gene products. The 20 initial positive clones (1 to 20) were inoculated into SD-TL and SD-TLHA medium and analyzed for lacZ reporter gene expression. The 13 clones (1–5, 7, 10–12, 16–18, and 20) can grow on SD-TLHA medium and turn blue in the β-galactosidase assay, indicating that these clones are real positive clones

Fig. 4

Retransformation validation of representative five host genes interacting with P1 Cap gene products in yeast two-hybrid system. The five clones (1, 3, 10, 16, and 18) were inoculated into SD-TL and SD-TLHA medium plates and analyzed for lacZ expression. The five clones that grew on SD-TLHA medium and turned blue in the yeast were positive. The images were cropped, and full-length images are included in Supplementary Fig. 4a to 4c

Fig. 5

Co-immunoprecipitation of Cap interacted with Ral GDS. HEK 293 Graham cells were co-transfected with pcDNA3.1-Ral GDS-FLAG and pcDNA3.1-His (C1); pcDNA3.1-Ral GDS-FLAG and pcDNA3.1-ORF1-His (T1); pcDNA3.1-FLAG and pcDNA3.1-ORF1-His (C2); and pcDNA3.1-Ral GDS-FLAG and pcDNA3.1-ORF1-His (T2). The immune complexes were incubated with anti-FLAG (A) or anti-His antibodies (B), and then pre-coupled to protein A agarose beads and subjected to Western blotting analysis. The gels/blots were cropped, and full-length blots/gels are presented in Supplementary Fig. 5a to 5d. The cropped Fig. 5 A comes from the Supplementary Fig. 5a and 5b; and B comes from the Supplementary Fig. 5c and 5d