Torque teno virus (SANBAN isolate) ORF2 protein suppresses NF-kappaB pathways via interaction with IkappaB kinases

Abstract

Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor kappaB (NF-kappaB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-kappaB activity in a dose-dependent manner in the canonical NF-kappaB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-kappaB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IkappaBalpha protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKbeta as well as IKKalpha, but not IKKgamma. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-kappaB activity in the noncanonical NF-kappaB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-kappaB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-kappaB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-kappaB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.

FIG. 1.

The TTV ORF2 protein suppresses NF-κB activation by TNF-α in a dose-dependent manner in HeLa, HepG2, and RAW264.7 cells. (A) A Western blot analysis detected the expression of the TTV ORF2 protein in HeLa (lanes 1 and 2), HepG2 (lanes 3 and 4), and RAW264.7 (lanes 5 and 6) cells. Cells were transfected with 0.8 μg of pcDNA3.1(−) as a control (lanes 1, 3, and 5) or with 0.8 μg of plasmid pcDNA3.1(−)-TTV2, expressing the TTV ORF2 protein (lanes 2, 4, and 6). At 48 h posttransfection, cell extracts were prepared and expression was determined using rabbit anti-TTV ORF2 antibody. (B) HeLa, HepG2, and RAW264.7 cells were cotransfected with 0.1 μg of pNF-κB-Luc and 10 ng of tk-Renilla-Luc along with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−) as a negative control. At 48 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for luciferase assay. (C) HeLa cells were cotransfected with 0.1 μg of pNF-κB-Luc and 10 ng of tk-Renilla-Luc along with different amounts of pcDNA3.1(−)-TTV2 (lane 3, 0 ng; lane 4, 200 ng; lane 5, 400 ng; lane 6, 600 ng). The total amount of plasmid was adjusted with the empty vector pcDNA3.1(−). At 48 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for the luciferase assay. Luciferase activities correspond to an average of results from at least three independent experiments, and data are shown as means ± SE (*, P < 0.05).

FIG. 2.

TTV ORF2 protein does not alter the expression of NF-κB but affects its nuclear translocation in HeLa cells. (A) HeLa cells were transfected with the empty vector pcDNA3.1(−) as a control (lanes 1 and 3) or with pcDNA3.1(−)-TTV2 (lanes 2 and 4). At 48 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 1 h (lanes 3 and 4), cell extracts were prepared and detected by Western blot analysis with anti-p65, anti-p50, and anti-β-actin antibodies. (B) HeLa cells were transfected with 0.8 μg of either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2. At 48 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 1 h, cytoplasmic and nuclear fractions were prepared as described in Materials and Methods. Both cytoplasmic proteins and nuclear proteins were analyzed by SDS-PAGE and Western blotting with anti-p65 and anti-p50 antibodies to reveal the localization of NF-κB subunits. Nucleus-specific anti-YY1 antibody and anti-GAPDH antibody were used as controls. Results are representative of three different experiments.

FIG. 3.

TTV ORF2 protein inhibits IκBα protein degradation. (A) HeLa cells were transfected with either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2. At 48 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 1 h or no stimulation as indicated, cell extracts were prepared and IκBα expression was determined by Western blotting using anti-IκBα antibody. β-Actin was used as a control. (B) HeLa cells were transfected with either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2. At 48 h posttransfection and after being treated with TNF-α (10 ng/ml) and the proteasome inhibitor MG-132 (20 μM) for the indicated times, cell extracts were prepared and phosphoserine IκBα levels were determined by Western blotting using anti-phosphoserine IκBα (P-IκBα) antibody. β-Actin was used as a control. Results are representative of three different experiments.

FIG. 4.

TTV ORF2 protein interacts with IKKα and IKKβ. HeLa cells were transfected with either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2. At 48 h posttransfection and after being treated with TNF-α (10 ng/ml) for 4 h, cell lysate was prepared for immunoprecipitation with monoclonal anti-IKKα, anti-IKKβ, or anti-IKKγ antibodies, with immunoglobulin G (IgG) taken as a control. Immune complex captured by protein A-Sepharose was separated by SDS-PAGE and analyzed by Western blotting with anti-TTV ORF2 antibody. One-tenth of the total cell lysates used for immunoprecipitation was loaded as a positive control for IKKα, IKKβ, and IKKγ. Results are representative of three different experiments. IP, immunoprecipitation; IB, immunoblotting.

FIG. 5.

The TTV ORF2 protein suppresses NF-κB activity in the noncanonical NF-κB pathway. (A) HeLa, HepG2, and RAW264.7 cells were cotransfected with 0.1 μg of pcDNA3.1(+)-NIK or pcDNA3.1(+)-dnNIK, 0.1 μg of pNF-κB-Luc, 10 ng of tk-Renilla-Luc along with 0.6 μg of pcDNA3.1(−)-TTV2, or 0.6 μg of pcDNA3.1(−) as indicated below the graph. At 48 h posttransfection, cells were harvested for luciferase assay. Luciferase activities correspond to averages of results from at least three independent experiments, and data are means ± SE (*, P < 0.05). (B) HeLa cells were transfected with 0.1 μg of pcDNA3.1(+)-NIK and 0.6 μg of either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2 as indicated below the graph. At 48 h posttransfection, cytoplasmic and nuclear fractions were prepared as described in Materials and Methods. Both cytoplasmic proteins and nuclear proteins were analyzed by Western blotting with anti-p100/p52 and anti-Rel-B antibodies to reveal the localization of NF-κB subunits. Nucleus-specific anti-YY1 antibody was used as a control. Results are representative of three different experiments.

FIG. 6.

TTV ORF2 protein inhibits the expression of NF-κB-mediated downstream genes through its effect on NF-κB. (A) HeLa and HEK293 cells were cotransfected with 0.1 μg of pGL3 X-p-Luc or pGL3 X-p-Luc (ΔNF-κB) (where X is COX-2, IL-6, or IL-8) and 10 ng of tk-Renilla-Luc along with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−) as indicated below the graph. At 48 h posttransfection, cells were treated with LPS (1 μg/ml) for 6 h and then harvested for luciferase activity. Luciferase activities correspond to averages from at least three independent experiments, and data shown are means ± SE (*, P < 0.05). (B) RAW264.7 cells were transfected with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−). At 48 h posttransfection, cells were stimulated with LPS (1 μg/ml) or left unstimulated for 6 h as indicated. The RT-PCR products of COX-2, IL-6, or IL-8 mRNA were detected by agarose gel electrophoresis. GAPDH was used as a control. IL-6, IL-8, and COX-2 mRNAs were measured by gel analysis software and normalized by calculating the ratio of mRNA to GAPDH. (C) RAW264.7 cells were transfected with 0.6 μg of the empty vector pcDNA3.1(−) without stimulation as a control or with different amounts of pcDNA3.1(−)-TTV2 (lane 2, 0 ng; lane 3, 200 ng; lane 4, 400 ng; lane 5, 600 ng) and treated with LPS (1 μg/ml) for 6 h. Cell extracts were prepared, and the protein amounts were determined using anti-COX-2 antibody. (D) RAW264.7 cells were transfected with 0.6 μg of pcDNA3.1(−)-TTV2 or 0.6 μg of pcDNA3.1(−). At 48 h posttransfection, cells were stimulated with LPS (1 μg/ml) or left unstimulated for 6 h as indicated below the graph. IL-6 and IL-8 in the culture supernatant of RAW264.7 cells were measured using the ELISA method. Results are representative of three different experiments.