Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability

Abstract

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions.

FIG. 1.

Tax1BP1 interacts with HPV16 and HPV18 E2 proteins. (A and B) C33A cells were transfected with expression vectors for FLAG-Tax1BP1 (TXBP) or control vector, along with either HA-tagged HPV18 E2 (A) or HPV16 E2 (B) expression plasmids. Cell extracts were immunoprecipitated (IP) with anti-FLAG (M2) or anti-EE control antibodies and immunoblotted (IB) with antibodies against HA (12CA5) (A) or HPV16 E2 (TVG261) (B). (C) Coimmunoprecipitation of endogenous Tax1BP1 with HPV16 E2. C33A cell extracts expressing HPV16 E2 were immunoprecipitated with an anti-Tax1BP1 antibody (TXBP151-C) or rabbit IgG and immunoblotted for HPV16 E2 (TVG261).

FIG. 2.

Tax1BP1 interacts with BPV1 E2. (A) Schematic diagrams of BPV1 E2 and Tax1BP1 constructs. Y2H, matched region in the initial yeast two-hybrid screen; WT, wild type. (B and C) C33A cells were transfected with expression vectors for FLAG-Tax1BP1 and the specified BPV1 E2 constructs. Reciprocal coimmunoprecipitations were performed by either immunoprecipitating (IP) BPV1 E2 (B201) and immunoblotting (IB) for FLAG-Tax1BP1 (M2) (B) or immunoprecipitating FLAG-TXBP (M2) and immunoblotting for E2 (II-1) (C). (D) C33A cells were transfected with expression vectors for FLAG-Tax1BP1 and E2 E39G, E2R, or control vector. Cell extracts were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted for E2 (II-1). (E) C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-tagged full-length Tax1BP1 (TXBP FL) or the truncation mutant TXBP-Nter or TXBP-Cter. Cell extracts were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted for E2 (II-1).

FIG. 3.

Tax1BP1 and p300 synergistically enhance E2-dependent transcription. The E2-dependent luciferase reporter was cotransfected into C33A cells with various expression plasmids. Luciferase activities were measured 24 h posttransfection and normalized against reporter alone. Each sample was analyzed in triplicate (the error bars indicate standard deviations). (A) Tax1BP1 and p300 synergistically enhance E2-dependent transcription. Expression vectors for BPV1 E2, HA-p300, and FLAG-Tax1BP1 alone or in combination were transfected as indicated. (B) The coactivator function of Tax1BP1 is p300 dependent. Expression vectors for BPV1 E2, FLAG-Tax1BP1, and adenovirus E1A 12S protein were transfected as indicated. (C) Tax1BP1 enhances E2 transcriptional activity in a dose-dependent manner. Expression vectors for BPV1 E2, HA-p300, and increasing amounts of FLAG-Tax1BP1 (125 ng, 500 ng, and 1 μg) were transfected. (D) Full-length Tax1BP1 is required for its coactivator function. Expression vectors for BPV1 E2 and FLAG-tagged full-length Tax1BP1 or the truncation constructs TXBP-Nter and TXBP-Cter were transfected.

FIG. 4.

Tax1BP1 interacts with p300 in vivo. C33A cells were transfected with expression vectors for FLAG-Tax1BP1 and HA-p300 or HA-luciferase control (HA-RLuc). Cell extracts were immunoprecipitated (IP) with anti-HA (12CA5) or anti-EE control antibodies and immunoblotted (IB) for FLAG-Tax1BP1 (M2).

FIG. 5.

E2 and Tax1BP1 are present at the BPV1 LCR. (A) A BPV1 genome-containing plasmid was cotransfected into C33A cells with expression vectors for BPV1 E2 and FLAG-Tax1BP1. Cell extracts were immunoprecipitated (IP) with mouse or rabbit antibodies (Ab) against FLAG, E2, or Tax1BP1 or control antibodies. The LCR of the BPV1 genome was amplified by PCR. (B) BPV1 genome-containing A3 cell extracts were immunoprecipitated with anti-E2 (II-1) or anti-Tax1BP1 (TXBP151-C) antibodies or rabbit IgG. The LCR of the BPV1 genome was amplified by PCR.

FIG. 6.

Tax1BP1 stabilizes E2 by preventing its proteasomal degradation. (A) Tax1BP1 increases the level of BPV1 E2 protein in a dose-dependent manner. C33A cells were transfected with expression vectors for BPV1 E2 and increasing amounts of FLAG-Tax1BP1. Cell extracts were resolved on SDS-PAGE and immunoblotted (IB) for FLAG-Tax1BP1 (M2), E2 (B201), or β-actin. (B and C) C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-Tax1BP1 or control vector. (B) At 24 and 48 h posttransfection, cell extracts were resolved on SDS-PAGE and immunoblotted for FLAG-Tax1BP1 (M2), E2 (B201), c-Myc (9E10), or β-actin. (C) At 24 and 48 h posttransfection, total RNA was extracted using TRIzol reagent. RNase-free DNase digestion of the isolated RNA was performed to remove the contamination of E2 plasmid DNA. E2 and GAPDH mRNAs were amplified by semiquantitative RT-PCR. PCR using increasing amounts of E2 plasmid DNA as templates were also performed as a control. (D) Tax1BP1 prevents proteasomal degradation of BPV1 E2. C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-Tax1BP1 or control vector. At 40 h posttransfection, the cells were treated with 10 μM MG132 or DMSO (mock) for 8 h. Cell extracts were resolved on SDS-PAGE and immunoblotted for FLAG-Tax1BP1 (M2), E2 (B201), or β-actin. (E) Tax1BP1 extends the half-life of BPV1 E2. C33A cells were transfected as described for panel D. At 40 h posttransfection, the cells were treated with cycloheximide (50 μg/ml) for up to 8 h. Cell extracts were resolved on SDS-PAGE and immunoblotted for FLAG-Tax1BP1 (M2), E2 (B201), or β-actin. (F) Graphical presentation of data presented in panel E. The level of E2 was quantitated and normalized using β-actin. (G) siRNA knockdown of endogenous Tax1BP1 reduces BPV1 E2 stability. C33A cells were transfected with control or Tax1BP1 siRNA and expression vectors for BPV1 E2. Total RNA was extracted. Tax1BP1 and GAPDH mRNAs were amplified by RT-PCR. Duplicated cell extracts were resolved on SDS-PAGE and immunoblotted for endogenous Tax1BP1 (TXBP151-C), E2 (B201), or β-actin.

FIG. 7.

The C-terminal region of Tax1BP1 stabilizes E2. (A) TXBP-Cter increases the level of BPV1 E2 protein in a dose-dependent manner. C33A cells were transfected with expression vectors for BPV1 E2 and increasing amounts of the FLAG-tagged Tax1BP1 truncation construct TXBP-Nter or TXBP-Cter. Cell extracts were resolved on SDS-PAGE and immunoblotted (IB) for FLAG-Tax1BP1 (M2), E2 (B201), or β-actin. (B) TXBP-Cter prevents proteasomal degradation of BPV1 E2 protein. C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-tagged TXBP-Nter, TXBP-Cter, or control vector. At 40 h posttransfection, the cells were treated with 10 μM MG132 or DMSO (mock) for 6 h and analyzed by Western blotting as described for Fig. 6D. (C) TXBP-Cter extends the half-life of BPV1 E2 protein. C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-tagged TXBP-Nter, TXBP-Cter, or control vector. At 40 h posttransfection, cycloheximide chase experiments were performed and analyzed as described for Fig. 6E. (D) Graphical presentation of data presented in panel C. The levels of E2 were quantitated and normalized using β-actin.

FIG. 8.

Tax1BP1 stabilizes the N-terminal TAD of BPV1 E2. C33A cells were transfected with expression vectors for E2TAD (A) or E2R (B) and FLAG-tagged full-length Tax1BP1, the truncation constructs TXBP-Nter and TXBP-Cter, or control vector. At 40 h posttransfection, the cells were treated with 10 μM MG132 or DMSO (mock) for 6 h and analyzed by Western blotting (IB) as described for Fig. 6D.

FIG. 9.

E2 stabilization by Tax1BP1 is independent of Tax1BP1 ubiquitin binding. (A) Ubiquitin-binding-defective Tax1BP1 mutants interact with BPV1 E2. C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-tagged full-length wild-type Tax1BP1 (WT); the ubiquitin-binding-defective mutants UBZ1, UBZ2, and UBZ*; or the truncation mutant TXBP-Nter or TXBP-Cter. To ensure equal levels of E2 inputs, the cells were treated with 10 μM MG132 for 6 h. Cell extracts were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted (IB) for E2 (II-1). (B) Ubiquitin-binding-defective Tax1BP1 mutants increase the level of BPV1 E2 protein. C33A cells were transfected with expression vectors for BPV1 E2 and FLAG-tagged wild-type Tax1BP1 (WT), UBZ1, UBZ2, UBZ*, or control vector. Cell extracts were resolved on SDS-PAGE and immunoblotted for FLAG-Tax1BP1 (M2), E2 (B201), or β-actin. (C) in vivo ubiquitination assay. C33A cells were transfected with expression constructs for BPV1 E2, FLAG-tagged full-length Tax1BP1, and HA-tagged ubiquitin. Forty hours posttransfection, the proteasome inhibitor MG132 dissolved in DMSO was added to the cells for 6 h at a final concentration of 10 μM. Cell extracts were immunoprecipitated with anti-E2 antibody (II-1) or rabbit IgG and immunoblotted for HA-ubiquitin (12CA5), FLAG-Tax1BP1 (M2), or E2 (B201).