Metabolic Labeling of Woodchuck Hepatitis B Virus X Protein in Naturally Infected Hepatocytes Reveals a Bimodal Half-Life and Association with the Nuclear Framework

Identification and stability of WHx in the cytosol of WHV-infected hepatocytes.

Our previous study utilized immunoprecipitation in combination with a Western blot-enhanced chemiluminescence (ECL) detection method to identify steady-state levels of WHx in the cytoplasmic fraction of hepatocytes isolated from woodchucks persistently infected with WHV (11). In an effort to increase the sensitivity of our detection system and to determine the turnover of WHx in naturally infected hepatocytes, cell fractionation procedures followed by immunoprecipitation of metabolically radiolabeled WHx were performed in this study. Primary hepatocytes were isolated by collagenase perfusion from adult woodchucks with persistent WHV infection and from uninfected animals (11, 17, 28). Hepatocytes (>90% viability) were seeded onto 10-cm tissue culture plates (4 × 10⁶ cells/dish) and maintained for 2 to 3 weeks, at 37°C in a 5% CO₂ atmosphere, in a previously described L-15 modified medium (1) supplemented with 5% fetal bovine serum. For metabolic radiolabeling, cells were preincubated for 30 min (37°C) in methionine- and cysteine-free RPMI 1640 (Gibco/BRL) supplemented with 5% dialyzed fetal bovine serum (Sigma), and then 300 μCi of ³⁵S-Promix (Amersham) per ml was added. After 2 h, the labeling medium was removed and the cells were chased with nonradioactive medium for various times. After the chase, hepatocytes were washed in ice-cold phosphate-buffered saline, harvested by scraping, and collected by low-speed centrifugation (50 × g, 5 min).

To answer the question of whether WHx is a soluble cytoplasmic protein or whether it is associated with other cytoplasmic constituents, ³⁵S-labeled hepatocytes were resuspended in 10 volumes of hypotonic TKM buffer (10 mM Tris [pH 7.5], 1 mM MgCl₂, 5 mM KCl, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1 mg of leupeptin per ml, 1 mg of pepstatin A per ml) and disrupted by Dounce homogenization. Hepatocyte breakage was monitored by phase-contrast microscopy. Nuclei, plasma membrane sheets, and remaining whole cells were first removed by low-speed centrifugation (1,000 × g, 10 min), and then the postnuclear supernatants were fractionated by high-speed centrifugation (100,000 × g, 2 h) into a soluble S100 fraction (cytosolic fraction) and a particulate P100 fraction (microsomal fraction). After the S100 supernatants were removed, the P100 pellets (microsomal fractions) were dissolved in detergent buffer (TKM buffer supplemented with 150 mM NaCl, 1% Nonidet P-40, and 1% sodium dodecyl sulfate [SDS]), passed through a 26-gauge needle, and then used for the immunoprecipitation with WHx-antiserum or normal rabbit serum as previously described (11). Similarly, equivalent amounts of S100 cytosolic proteins were used for the immunoprecipitation. Immunoprecipitates were finally solubilized in SDS-Laemmli sample buffer, resolved on a 13% SDS-polyacrylamide gel electrophoresis (PAGE), and visualized by autoradiography.

As shown in Fig. 1A, ³⁵S-labeled WHx was specifically immunoprecipitated with the rabbit WHx antiserum from the S100 cytosolic fraction of WHV-positive hepatocytes (S100 lanes 3 and 4), while no protein of similar size (15.5 kDa) was immunoprecipitated from uninfected woodchuck hepatocytes (lane 1). Normal rabbit serum also did not immunoprecipitate a 15.5-kDa protein from the same WHV-infected cell extracts (lane 2). A quantitative evaluation of the amount of labeled WHx present in the S100 fraction after 0 and 30 min of chase (Fig. 1A, S100 lanes 3 and 4, respectively) indicated that cytosolic WHx has a short half-life in chronically infected hepatocytes, since its amount was reduced to 45% after 30 min of chase.

³⁵S-labeled WHx was not detected in immunoprecipitates from P100 particulate fractions (Fig. 1A), which contained fragments of plasma membrane, intracellular membranes such as the endoplasmic reticulum and the Golgi, and organelles such as lysosomes and mitochondria (32). The distribution of protein disulfide isomerase (43), a specific marker for the endoplasmic reticulum, was analyzed to confirm the purity of the cytosolic and microsomal fractions. Protein disulfide isomerase was exclusively detected by immunoblotting in the P100 microsomal fraction (data not shown). These findings indicate that WHx is not tightly associated with these subcellular compartments.

To determine the kinetics of turnover of cytosolic WHx, cells were metabolically radiolabeled for 1 h and chased up to 60 min (Fig. 1B). The S100 cytosolic fractions, containing equal amounts of total proteins, were immunoprecipitated with WHx antiserum (Fig. 1B, lanes 2 to 8). Densitometric analysis of the fluorographs of immunoprecipitated WHx revealed a bimodal half-life of cytosolic WHx in the first hour of chase. In fact, approximately 50% of labeled WHx was no longer detected at 15 to 20 min of chase. In contrast, the remaining WHx turned over more slowly, since 40% of metabolically radiolabeled WHx was still detected after 1 h of chase (Fig. 1C). Longer chase experiments, extending up to 6 h, also confirmed the presence of the more stable WHx component (see below).

Subcellular distribution of labeled WHx.

A bimodal degradation of newly synthesized proteins suggested that compartmentalization and/ or posttranslational modifications of WHx might have occurred. To investigate whether a different compartmentalization of WHx could account for the bimodal turnover observed in the cytoplasm, we next used a differential detergent extraction procedure to fractionate hepatocytes chronically infected with WHV (36, 42). Cells were labeled for 1 h with ³⁵S-Promix (300 μCi/ml), harvested as described above, and then resuspended in TX buffer (10 mM Tris [pH 7.5], 250 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 2 mM CaCl₂, 0.5% Triton X-100, 2 mM PMSF, 1 mg of leupeptin per ml, 1 mg of pepstatin A per ml). After an 8-min incubation on ice, the cells were centrifuged (600 × g, 3 min) to pellet the Triton-insoluble fraction. As highlighted in Fig. 2A, this procedure first separated soluble cytoplasmic and nucleoplasmic proteins (the TX-sol fraction) from Triton-insoluble cellular components (P1). Next, the P1 pellet was resuspended in CSK buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 1.5 mM MgCl₂, 1% Tween 40, 0.5% Na deoxycholate, 2 mM PMSF, 1 mg of leupeptin per ml, 1 mg of pepstatin per ml) to solubilize the cytoskeletal components. The solution was passed through a 26-gauge needle 20 times, and then the CSK fraction was separated by centrifugation (1,000 × g for 5 min) from the insoluble nuclear framework fraction (P2). Finally, the proteins in the nuclear framework pellet, which is mainly composed of nuclear matrix and chromatin (5, 42), were resuspended in CSK buffer supplemented with 1% SDS and passed through a 26-gauge needle to be solubilized (NM fraction).

To determine the subcellular distribution of labeled WHx in the TX-sol, CSK, and NM fractions, we conducted immunoprecipitation-PAGE with either WHx antiserum (Fig. 2B, lanes 2) or rabbit normal serum (Fig. 2B, lanes 1). Quantitative analysis of the amount of labeled WHx recovered from each fraction revealed 70 to 75% of labeled WHx in the TX-sol fraction, 5 to 10% in the CSK fraction, and, to our surprise, approximately 20% in the NM fraction (Fig. 2C).

To check the efficiency of this cell fractionation procedure, the NM fraction was assayed for the presence of contaminant cytoplasmic proteins. Figure 3A shows that ligandin, a cytosolic protein involved in bilirubin intracellular transport (50), was completely extracted into the TX-sol fraction, as determined by SDS-PAGE and ECL-Western blotting with specific antiligandin antibodies (gift from I. Listowsky, Albert Einstein College of Medicine, Bronx, N.Y.). Furthermore, aliquots from each cell fraction were analyzed by SDS-PAGE and Coomassie blue staining. As shown in Fig. 3B, the histone proteins were retained entirely in the NM fraction. Protein concentration analyses and Coomassie blue staining (Fig. 3B) also demonstrated that the CSK and the NM fractions contained much smaller amounts of total proteins (about 15 and 10%, respectively) than the TX-sol fraction (approximately 75 to 78%). Therefore, the Triton-insoluble fractions were considerably enriched for WHx.

Our results are consistent with those of Schek et al. (36), who previously described a similar subcellular distribution and bimodal half-life of HBx in HepG2 cells when vaccinia virus vectors were used to induce transient overexpression of HBx protein. A similar partition of WHx between soluble and cytoskeletal fractions has also been observed recently in hepatocytes isolated from acutely WHV-infected woodchucks, although nucleus-associated WHx was not detected under the conditions used in that study (20). However, another report by Doria et al. (12) provided evidence, by confocal laser microscopy, for the presence of a small fraction of HBx within the nuclei of transfected cells. It is possible that some discrepancies in terms of X-protein distribution and solubility might be due to differences in the techniques used as well as differences in the expression levels of X protein obtained after infection with recombinant systems (12, 36, 44) versus the low steady-state levels of WHx actually present in naturally infected hepatocytes.

Half-life of TX-sol WHx and kinetics of association of labeled WHx with the nuclear framework.

To examine the rate of association of WHx with the NM fraction and to determine the half-lives of WHx in the TX-sol and Triton-insoluble fractions, WHV-positive hepatocytes were pulse-labeled for a short time (10 min) with 600 μCi of ³⁵S-Promix per ml at 37°C. Two 10-cm plates of confluent woodchuck hepatocytes were used per time point. After the chase, cells were washed in ice-cold phosphate-buffered saline and fractionated by using the differential detergent extraction procedure outlined in Fig. 2A. TX-sol fractions containing the same amount of total proteins per chase point were immunoprecipitated with WHx antiserum, and immunoprecipitates were analyzed by SDS-PAGE. Quantitative analyses of the autoradiographs shown in Fig. 4A (chase up to 1 h) and Fig. 4B (chase up to 6 h) demonstrated that the turnover of TX-sol WHx was bimodal. In fact, within 20 min of chase, 50% of TX-sol WHx turned over while the remaining 50% decayed with a half-life of 3 to 4 h (Fig. 4C).

Densitometric scanning of the NM data showed that newly synthesized WHx rapidly associated with the NM fraction (Fig. 4A, NM lane 2), since it reached a maximum immediately after the pulse (0 min of chase). Densitometry was difficult to analyze because of the weakness of the signal after only 10 min of pulse, and the amount of labeled WHx in the cytoskeletal fractions was below the limit of detection (data not shown). However, results of several experiments (one of which is shown in Fig. 4A) showed that WHx did not accumulate within the NM fraction, where it appeared to have a short half-life (approximately 30 min). Therefore, our data support the hypothesis that subcellular distribution of WHx may be responsible, at least in part, for the bimodal kinetics of WHx observed in the cytoplasm.

The cytoplasmic cytoskeleton is contiguous with the nuclear matrix (5), and several studies have shown that these structures may support the transport of viral components to the nucleus (31, 35, 42). The presence of small amounts of WHx associated with the CSK fraction would suggest this possibility. However, it is also possible that WHx could enter the nucleus by diffusion, since the X protein is smaller than the exclusion limit of the nuclear pore. Nuclear WHx could then be retained in the nucleus by binding to a nondiffusible nuclear component, like the nuclear matrix. Our finding suggests that a fraction of newly synthesized WHx actually associates with components of the nuclear framework and that the transit from the soluble fraction to the insoluble framework fraction is rapid. This would also explain why we did not detect WHx in nucleoplasmic extracts when standard procedures to detect unlabeled WHx were used (11).

Overall, our subcellular fractionation data indicate that the great majority of WHx is a soluble cytoplasmic protein and that only a minor fraction of WHx is associated with nuclear components in vivo. An accumulating body of evidence (3, 9, 10, 12, 21, 24, 27) suggests that the cytosol is an important site for X-protein transactivating function. Thus, the presence of WHx in the cytosol of chronically infected hepatocytes might lead to a moderate but constitutive activation of several components of the cytoplasmic signaling cascades.

A short half-life is a typical feature of many viral and cellular proteins that have transcriptional regulatory functions, such as the Tax protein of human T-cell leukemia virus type 1 (40), the E1A protein of adenovirus (41), and the c-Myc protein (47). Furthermore, the presence of newly synthesized WHx associated with the NM fraction gives rise to the idea of a possible functional interaction between nuclear transcription factors and structural components of the nuclear matrix. It is known that the nuclear matrix plays an important role in regulating gene expression, and it has been shown that several transcription factors, like the ubiquitous transcription factor YY1 (15), ATF1 (16), and Oct-1 (23), as well as the retinoblastoma gene product Rb (2), the glucocorticoid receptor (13), the simian virus 40 large T antigen (37), and the E1A herpesvirus protein (6), are partially sequestered in the nuclear matrix. Studies targeted towards investigating such interactions may shed further light on the natural mechanism of action of WHx in hepatocytes.