Different regions of hepatitis B virus X protein are required for enhancement of bZip-mediated transactivation versus transrepression

Abstract

The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIgamma and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIgamma interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX(49-115) is as effective as the full-length pX in enhancing the ATF3- and ICERIIgamma-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX(49-140) are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.

FIG. 1

(A) Diagram of constructed pX deletion mutants. pX regions A, B, and C are conserved to various degrees in hepadnaviruses and are essential for transactivation. C₆₁ and C₆₉ are conserved cysteines. The polymerase II, RPB5 subunit, TFIIH, and TFIIB interacting regions are indicated. (B) Coomassie blue-stained SDS gel (14%) of purified (4), recombinant GST-X and the indicated pX deletions. •, GST-X fusion protein; ∗, endogenously cleaved GST portion. (C) Western blot of recombinant wt pX, pX_49–154, pX_79–154, pX_49–140, pX_49–115, and pX_49–90 (GST fusions) detected with a polyclonal pX-specific antibody. •, GST-X fusion protein. Protein bands smaller than the GST-X fusion proteins are degradation products.

FIG. 2

K_d determination of CREB-pX interaction. GST-X_1–154 resin (⧫) (5 μg) was incubated with increasing amounts of ³²P-CREB (0.2 to 62.5 nM) for 3 h at 4°C in buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, 0.1% Triton X-100, 5 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, and 50 μg of BSA per ml. The beads were washed six times and eluted by boiling in SDS-PAGE loading buffer. Analysis was done on SDS–12% PAGE gels. Equivalent amounts of GST-bound resin (■) or equal amounts of glutathione beads (▴) were used as the control. Bound CREB was plotted against the total concentration of CREB. The K_d value was calculated as the concentration of CREB at which half-maximal binding to GST-X was observed. The experiment was repeated three times and shows a K_d value of approximately 1.8 × 10⁻⁸ M. A representative experiment from three independent experiments is shown above.

FIG. 3

Activity of pX deletions in vitro. (A) Protein-protein interaction assay with equivalent amounts of GST-X deletion mutants (3 μg) incubated with ³²P-CREB (5 to 10 ng) for 3 h at 4°C in buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, 0.1% Triton X-100, 5 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, and 50 μg of BSA per ml. The beads were washed six times and eluted by boiling in SDS-PAGE loading buffer. Analysis was done by SDS-PAGE on 12% gels. A total of 12 to 15 μg of GST bound to resin was used as a control. (B) Quantitation of relative CREB binding to each GST-X deletion mutant compared to wt pX carried out by scintillation counting of the gel excised bands. Nonspecific binding of CREB to the GST-resin control is subtracted from all of the plotted values. The data shown are from four independent experiments. (C) DNA-protein binding assays of recombinant CREB in the presence of pX mutants were carried out as described previously (3) with 10 ng of recombinant CREB and 10,000 cpm of radiolabeled CRE, with an equivalent amount of the following: + lanes, GST-X_49–140, GST-X_49–115, and GST-X_49–90; − lanes, recombinant GST protein, as indicated. Lanes containing GST-X_49–90 were exposed for 2 days at −80°C during autoradiography, as opposed to 18 h of exposure for the other lanes.

FIG. 4

Activity of pX deletions in vivo. (A) Mammalian two-hybrid assay of pX deletion mutants in AML12 cells. A total of 500 ng of Gal4_UAS-luciferase reporter was cotransfected by the FuGENE transfection protocol, with 700 ng each of RSV-CREB-VP16 and RSV-X-Gal4 expression vectors per 30-mm culture dish, performed in triplicate. Cells were harvested 18 h after transfection, and luciferase activity was quantitated and expressed per microgram of protein extract as previously described (3). Results are from three independent experiments. (B) Transient transfection of HBV pX mutants in AML12 cells. A total of 3 to 5 μg of CRE³-CAT reporter plasmid was cotransfected with pCMV-X-NLS (20 ng), pCMV-X_49–140 (100 ng), and pCMV-X_49–115 (50 ng) expressor plasmid. Control transfections contained equivalent amounts of pCMV-empty vector. The optimal amounts of pCMV-X expression vectors shown (wt and mutants) were established by titration analyses. Transfected cells were treated with 10 μM forskolin and 100 μM IBMX and harvested at 24 h. CAT assays were performed as described earlier (3). Relative CRE³-CAT induction values per microgram of protein extract were quantitated from three independent experiments.

FIG. 5

Interaction of pX deletions with ATF3 and ICERIIγ. (A) Protein-protein interaction assays of GST-X, GST-X_49–140, GST-X_49–115, and GST-X_49–90 with in vitro-translated, [³⁵S]methionine-radiolabeled ATF3 (3 μl) and ICERIIγ (7 μl) as indicated. Experimental conditions were as described in Fig. 3A. (B) Quantitation of relative ATF3 and ICERIIγ binding to each GST-X deletion mutant compared to wt pX as determined by scintillation counting of the gel-excised bands. Nonspecific binding of ATF3 and ICERIIγ to the GST-bound resin is subtracted from all of the plotted values. The data shown are from three independent experiments.

FIG. 6

Transient transfection of HBV pX mutants in AML12 cells in the presence of bZip repressors ATF3 and ICERIIγ. (A) A total of 600 ng of CRE-luciferase reporter plasmid was cotransfected by the FuGENE protocol (Boehringer Mannheim) with 100 ng of pCMV-X-NLS, pCMV-X_49–140, and pCMV-X_49–115 expressor plasmid in the presence of 50 ng of ATF3 expression vector. (B) CRE³-CAT reporter plasmid (5 μg) cotransfected by the calcium phosphate protocol (BRL) with pCMV-X-NLS (20 ng), pCMV-X_49–140 (50 ng), and pCMV-X_49–115 (50 ng) expressor plasmid in the presence of 0.5 μg of ICERIIγ. Optimal amounts of the indicated expression vectors transfected in AML12 cells were determined by titration analyses. Relative CRE³-CAT activities per microgram of protein extract were quantitated from three independent experiments.

FIG. 7

Model depicting concomitant interactions of pX with bZip transcription factors and the basal transcriptional apparatus. (A and B) pX-mediated CREB transactivation effected by pX_49–140 (containing regions A, B, and C) and pX_49–115 (containing regions A and B), respectively. (C and D) pX-mediated ICERIIγ transrepression effected by pX_49–140 and pX_49–115, respectively.