Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework

Abstract

In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.

FIG. 1

Localization and turnover of WHx in the cytosol of hepatocytes chronically infected with WHV (A) Woodchuck hepatocytes were metabolically radiolabeled with ³⁵S-Promix for 2 h and then chased with cold medium. After 0 (lanes 1 to 3) or 30 (lanes 4) min of chase, cell-equivalent amounts (6 × 10⁶ hepatocytes/assay) were fractionated into soluble (S100) and particulate (P100) cytoplasmic fractions, and fractions were immunoprecipitated with WHx antiserum (lanes 1, 3, and 4) or normal rabbit serum (lanes 2). Immunoprecipitates from uninfected (lanes 1) and from WHV-positive hepatocytes (lanes 2, 3, and 4) were then analyzed by SDS-PAGE (12%) and detected by autoradiography (2 weeks’ exposure). WHx was not detected in P100 fractions even after longer exposure of the gel (5 weeks). (B) Cells were labeled for 1 h and chased for the indicated periods (in minutes). Equivalent amounts of total soluble proteins (S100 fractions) per time point were immunoprecipitated with WHx antiserum (lanes 2 to 8) or with normal rabbit serum (lane 1) and fractionated by SDS-PAGE (13%). (C) Kinetics of turnover of cytosolic WHx. The amount of labeled WHx present in the S100 fraction at each time point was quantitated by scanning densitometry and normalized to the amount present at time zero (arbitrary scale).

FIG. 2

Subcellular distribution of labeled WHx protein in the TX-sol, CSK, and NM fractions. (A) Outline of the differential detergent extraction procedure used to investigate the subcellular distribution of WHx protein in primary hepatocytes isolated from woodchucks persistently infected with WHV. (B) Hepatocytes (6 × 10⁶) from a WHV-positive woodchuck were metabolically radiolabeled with ³⁵S-Promix for 1 h, harvested, and fractionated by differential detergent extraction. Fifty percent of each fraction was then immunoprecipitated with normal rabbit serum (lanes 1) or with WHx antiserum (lanes 2), and the immunoprecipitates were resolved on a SDS-PAGE (12%). (C) The amount of labeled WHx recovered from each fraction was quantitated by densitometry scanning of fluorographs. Each column represents the relative amount of labeled WHx detected in each fraction. These values have been reproduced in three independent experiments.

FIG. 3

Distribution of cellular marker proteins in the TX-sol, CSK, and NM fractions. (A) Aliquots representing 0.5% (vol/vol) of the TX-sol and 2% (vol/vol) of the NM fractions were analyzed by SDS-PAGE (13%) and subjected to ECL-Western blot analysis with an antiligandin rabbit antiserum. (B) Total proteins from the TX-sol (0.5%), CSK (1%), and NM (1%) fractions were also subjected to SDS-PAGE (13%) and Coomassie blue staining.

FIG. 4

Kinetics of the turnover of WHx protein in TX-sol and NM fractions. (A) WHV-infected hepatocytes were pulse-labeled with ³⁵S-Promix for 10 min and chased with cold medium for various times as indicated (in minutes). After 0 min and each chase time, 6 × 10⁶ cells per time point were fractionated as described in the text and in Fig. 2. Fractions were immunoprecipitated with WHx-antiserum (lanes 2 to 5) or with normal rabbit serum (lanes 1), subjected to SDS-PAGE (12%), and visualized by fluorography. (B) Metabolically radiolabeled WHV-positive hepatocytes were chased for longer periods, and immunoprecipitates from TX-sol fractions were analyzed by SDS-PAGE and autoradiography. (C) The amount of WHx present in the TX-sol fraction at each time point was quantitated by scanning densitometry and normalized (on an arbitrary scale) to the amount present at time zero. (D) The amount of labeled WHx associated with the NM fraction in the first hour of chase was normalized to the amount of labeled WHx present at time zero.