DDB2 induces nuclear accumulation of the hepatitis B virus X protein independently of binding to DDB1

Abstract

The hepatitis B virus (HBV) X protein (HBx) is critical for the life cycle of the virus. HBx associates with several host cell proteins including the DDB1 subunit of the damaged-DNA binding protein DDB. Recent studies on the X protein encoded by the woodchuck hepadnavirus have provided correlative evidence indicating that the interaction with DDB1 is important for establishment of infection by the virus. In addition, the interaction with DDB1 has been implicated in the nuclear localization of HBx. Because the DDB2 subunit of DDB is required for the nuclear accumulation of DDB1, we investigated the role of DDB2 in the nuclear accumulation of HBx. Here we show that expression of DDB2 increases the nuclear levels of HBx. Several C-terminal deletion mutants of DDB2 that fail to bind DDB1 are able to associate with HBx, suggesting that DDB2 may associate with HBx independently of binding to DDB1. We also show that DDB2 enhances the nuclear accumulation of HBx independently of binding to DDB1, since a mutant that does not bind DDB1 is able to enhance the nuclear accumulation of HBx. HBV infection is associated with liver pathogenesis. We show that the nuclear levels of DDB1 and DDB2 are tightly regulated in hepatocytes. Studies with regenerating mouse liver indicate that during late G1 phase the nuclear levels of both subunits of DDB are transiently increased, followed by a sharp decrease in S phase. Taken together, these results suggest that DDB1 and DDB2 would participate in the nuclear functions of HBx effectively only during the late-G1 phase of the cell cycle.

FIG. 1

DDB2 overexpression stimulates nuclear accumulation of HBx. A plasmid expressing HBx (5 μg) was transfected into U2OS cells either with empty vector or with a plasmid(s) expressing either T7-tagged DDB1 (5 μg), T7-tagged DDB2 (5 μg), or both. Crude cytosolic and nuclear extracts were prepared as described in Materials and Methods. Portions (100 μg) of the extracts were analyzed by Western blot assays. Blots were probed with polyclonal antibodies against HBx (3) as described in Materials and Methods. cdk2 levels were assayed as a loading control.

FIG. 2

The WD motif in DDB2 is important for binding to HBx. U2OS cells were cotransfected with FLAG epitope-tagged HBx plasmids and either T7-tagged wild-type DDB2 or a T7-tagged deletion mutant of DDB2 (1–380, 1–320, 1–260, or WDΔ). (Top) Total cell extracts of transfected cells were subjected to immunoprecipitation with T7 antibody conjugated to beads, followed by a Western blot assay. The blot was probed with antibodies against HBx (3). Co-IP, coimmunoprecipitate. (Center and bottom) To assay for expression of the HBx and DDB2 proteins, extracts were subjected to Western blot analysis using antibodies against HBx and the T7 epitope, respectively.

FIG. 3

The C-terminal region of DDB2 is essential for binding to DDB1. U2OS cells were transfected with V5-tagged DDB1 expression plasmids and either T7-tagged wild-type DDB2 or a T7-tagged C-terminal deletion mutant of DDB2 (1–380, 1–320, 1–260, 1–200, or WDΔ). (Top) Extracts of the transfected cells were subjected to immunoprecipitation using T7 antibodies. Immunoprecipitates were analyzed by a Western blot assay. The blot was probed with horseradish peroxidase-linked V5 antibody (Invitrogen) to detect coprecipitating DDB1. Co-IP, coimmunoprecipitate. (Center and bottom) Extracts were also tested for expression of DDB1 and DDB2 by probing the blots with V5 and T7 antibodies, respectively. The star indicates a nonspecific band.

FIG. 4

DDB2 overexpression facilitates nuclear accumulation of HBx in U2OS cells. U2OS cells were grown on plates containing coverslips and were transfected with plasmids (5 μg) expressing FLAG-tagged HBx either alone or in combination with either T7-tagged wild-type DDB2 or the indicated T7-tagged mutant. Coverslips containing transfected cells were fixed with methanol and probed with FITC-conjugated monoclonal antibodies against the FLAG epitope as described in Materials and Methods. Immunofluorescence was detected, and images were taken, using a CLSM 510 microscope. Panels on the left (L) represent immunofluorescing cells; panels on the right (R) represent their overlap with the respective phase-contrast micrograph.

FIG. 5

DDB2 overexpression enhances nuclear accumulation of HBx in HepG2 cells. HepG2 cells were grown on plates containing coverslips and were transfected with plasmids (5 μg) expressing FLAG-tagged HBx either alone or in combination with either T7-tagged wild-type DDB2 or the indicated T7-tagged mutant. Cells on coverslips were fixed and probed with FITC-conjugated monoclonal antibodies against the FLAG epitope. Immunofluorescence was detected, and images were taken, using a CLSM 510 microscope. Panels on the left (L) represent immunofluorescing cells; panels on the right (R) represent their overlap with the respective phase-contrast micrograph.

FIG. 6

Hepatocytes exhibit elevated expression of the DDB subunits in nuclei following liver injury. Two-month-old wild-type CD-1 mice were subjected to a two-thirds hepatectomy. Regenerating livers were harvested at the indicated times following PHx, and portions of liver tissue were fixed in 4% paraformaldehyde, paraffin embedded, sectioned with a microtome, and used for immunohistochemical staining with affinity-purified peptide antibodies specific for DDB1 and DDB2 as described in Materials and Methods.

FIG. 7

Changes in levels of DDB proteins in regenerating mouse liver nuclear extracts. After PHx, mice were sacrificed at the indicated time points, and regenerating livers were harvested. Livers were washed with polyamine buffer and minced with a razor blade, and nuclear extracts were prepared as described in Materials and Methods. Five-hundred-microgram portions of the nuclear extracts were assayed for DDB1, DDB2, and cdk2 using the respective antibodies as described in Materials and Methods.