Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells

Abstract

DNA sequences containing CpG motifs are recognized as immunomodulators in several species. Phosphodiester oligodeoxyribonucleotides (ODNs) representing sequences from the genome of porcine circovirus type 2 (PCV2) have been identified as potent inducers (ODN PCV2/5) or inhibitors (ODN PCV2/1) of alpha interferon (IFN-alpha) production by porcine peripheral blood mononuclear cells (poPBMCs) in vitro. In this study, the IFN-alpha-inducing or -inhibitory activities of specific phosphodiester ODNs were demonstrated to be dependent on their ability to form secondary structures. When a poly(G) sequence was added to a stimulatory self-complementary ODN, high levels of IFN-alpha were elicited, and the induction was not dependent on pretreatment with the transfecting agent Lipofectin. In addition, the IFN-alpha-inducing ODN required the presence of an intact CpG dinucleotide, whereas the inhibitory activity of ODN PCV2/1 was not affected by methylation or removal of the central CpG dinucleotide. Of particular significance, the IFN-alpha inhibition elicited by ODN PCV2/1 was only effective against induction stimulated by DNA control inducers and not RNA control inducers, indicating activity directed to TLR9 signaling. The PCV2 genome as a whole was demonstrated to induce IFN-alpha in cultures of poPBMCs, and the presence of immune modulatory sequences within the genome of PCV2 may, therefore, have implications with regard to the immune evasion mechanisms utilized by PCV2.

FIG. 1.

Predicted secondary structure formation of ODNs. (a) Spontaneous self-dimer formation of ODNs H1a, b, and c; H2a, b, and c; H3b and c; and 2216 and the double-stranded formation generated after hybridization of ODNs H^Gtail2 and I^Gtail2. (b) Spontaneous hairpin formation of ODN PCV2/1 and its derivatives PCV2/1a, b, and c. The double-stranded conformations were predicted using the IDT SciTools Oligo Analyzer 3.0 software. In all cases, the conformation with the lowest ΔG value (kcal/mol) is given.

FIG. 2.

Inhibitory effect of ODN PCV2/1 and its derivatives PCV2/1a, b, and c on IFN-α production induced by the control ODN 2216, ADV, plasmid DNA (pcDNA3), poly(I:C), or SV. The IFN-α production is expressed as the percentage of inhibition of the IFN-α produced in cultures induced with the controls alone. The results are given as mean values ± 95% confidence intervals so that nonoverlapping confidence intervals indicate statistically significant differences (n ≥ 6). All derivatives of ODN PCV2/1 were used at a final concentration of 25 μg/ml. ODN 2216 and poly(I:C) were used at 5 μg/ml, and pcDNA3 was used at 2.5 μg/ml. ADV was used at a concentration corresponding to 10³ 50% infectious doses per ml before UV inactivation, and SV was diluted 1:10.

FIG. 3.

Effect of ODN PCV2/1 on the frequency of IFN-α-producing cells in cultures of poPBMCs induced with the control ODN 2216, ADV, or poly(I:C). The number of IFN-α-producing cells was determined by ELISPOT assay in cultures induced by the controls alone (solid lines) or in combination with ODN PCV2/1 (dashed lines). The spot frequency was determined at three cell concentrations, 0.5 × 10⁶, 1 × 10⁶, and 2 × 10⁶ cells/ml (pig no. 1, closed circles; pig no. 2, closed squares; pig no. 3, closed triangles wide base; pig no. 4, closed triangles narrow base; pig no. 5, open circle; pig no. 6, open squares).

FIG. 4.

Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.