Specific association of endogenous and ectopically expressed SRPK1 and SRPK2 with GST-HBV core protein. COS-7 cells were transiently transfected with either control vectors or plasmids encoding FLAG-tagged SRPK1 (1.5 μg/well) or VSV-tagged SRPK2 (1.0 μg/well). After 24 h, cells were lysed, and extracts were subjected to in vitro association with either GST or GST-HBV-C1 protein. Samples were then resolved in duplicate by SDS-PAGE, followed by immunoblotting with anti-SRPK1 and anti-FLAG antibodies (left panels) or anti-SRPK2 and anti-VSV antibodies (right panels).

Detection of specifically associating HBV core protein kinases by an in-gel kinase assay. Total cell lysates prepared from HuH-7 cells were subjected to in vitro association with either GST or different GST-HBV core fusion proteins containing N-terminally truncated HBV core protein (GST-HBV-C1 and GST-HBV-C2) or wild-type HBV core protein (GST-HBV-C3). Bound proteins eluted with SDS sample buffer and total cell lysate were then resolved in parallel on gels containing either copolymerized GST-HBV-C1 (left panel) or GST protein (right panel). Upon denaturation and renaturation, in-gel kinase reactions were performed in the presence of [γ-³²P]ATP. Gels were then washed and subjected to autoradiography.

Comparison of SRPK1- and SRPK2-mediated HBV core protein phosphorylation. HuH-7 cells were infected with adenovirus as described for Fig. 9A. Total cellular extracts were assayed for HBV core kinase activity in an in vitro kinase assay with GST-HBV core protein C1 as a substrate. After gel electrophoresis, phosphate incorporation into substrate protein was visualized by autoradiography (upper panel). In parallel, identical samples were analyzed by immunoblotting with either anti-SRPK1, anti-SRPK2, or anti-VSV antibodies (lower three panels). Positions of kinases and kinase substrates are indicated on the right.

Cellular PKC activity does not mediate HBV core protein phosphorylation. Total cell lysates from HuH-7 cells were incubated with either dimethyl sulfoxide or the indicated amounts of bisindolylmaleimide I (Bis-1). Aliquots were then added to kinase reaction mixes containing the same concentrations of bisindolylmaleimide I, which were supplemented with calcium, phosphatidylserine (PS), and diacylglycerol (DAG) where indicated. Kinase reactions were performed in the presence of either histone H1 (upper panel) or GST-HBV-C1 protein (lower panel). After gel electrophoresis, substrate phosphorylation was visualized by autoradiography. Positions of kinase substrates are indicated on the right.

Cyclin-dependent kinases Cdc2 and Cdk2 are not major cellular HBV core protein kinases. HuH-7 cells were either mock treated or incubated for 17 h in the presence of 0.25 μg of nocodazole per ml prior to lysis. Total cellular extracts were then incubated without beads or in the presence of either p9^CKShs1-agarose or control beads. Supernatants were then assayed for either histone H1 or HBV core kinase activity. After gel electrophoresis, phosphate incorporation into histone H1 or GST-HBV-C1 was visualized by autoradiography (first and fourth panel from top, respectively). In parallel, identical samples were analyzed by immunoblotting with either anti-Cdc2, anti-Cdk2, anti-SRPK1, or anti-SRPK2 antibodies. Positions of kinases and kinase substrates are indicated on the right.

Phosphorylation of GST-HBV core protein by associating cellular kinases. Total cell lysates prepared from HuH-7 cells were incubated with either GST or different GST-HBV core fusion proteins. GST-HBV-C1 and GST-HBV-C2 are GST fusion proteins with N-terminally truncated core proteins that start at amino acid positions 119 and 30, respectively. GST-HBV-C3 contained wild-type HBV core protein. After in vitro association for 2.5 h, kinase reactions were performed on the washed beads containing bound GST fusion proteins as substrates for interacting cellular kinases. Samples were then resolved by SDS-12.5% PAGE. Upon Coomassie staining to visualize the different GST fusion proteins (lower panel), autoradiography was performed to detect the phosphorylated GST-HBV core fusion proteins (upper panel). Positions of GST fusion proteins are indicated on the right. In this and subsequent figures, sizes are shown in kilodaltons.

Purification of specifically associating GST-HBV core protein kinases. Total cell extracts from HuH-7 cells were incubated with either GST or GST-HBV-C1 immobilized on glutathione-Sepharose beads. After in vitro association, bound proteins were then resolved by 16-BAC-7.5% PAGE in the first dimension, and the two lanes containing the GST and GST-HBV-C1 in vitro association samples were then resolved on the same 10% SDS gel containing copolymerized GST-HBV-C1 in the second dimension as described under Materials and Methods. Upon denaturation and renaturation, an in-gel kinase reaction was performed in the presence of [γ-³²P]ATP. Gels were then washed, Coomassie stained, and subjected to autoradiography. The arrows indicate the position of the faster-migrating HBV core protein kinase in the upper panel and its corresponding protein spot visualized by Coomassie staining in the lower panel. For subsequent mass spectrometry analysis, the same spot was excised from 16-BAC/SDS gels containing no copolymerized GST-HBV-C1 protein as described under Materials and Methods.

Isolation of stable HBV core protein-SRPK2 complexes from HuH-7 cells. (A) HuH-7 cells were infected in six-well dishes with adenovirus at 5,000 particles of AdHBVcore per cell plus either control adenovirus (no expression cassette), 25,000 particles of AdSRPK1-VSV per cell, or 15,000 particles of AdSRPK2-VSV per cell as indicated. Control adenovirus was added to give a total particle number of 30,000/cell for each of the infections. On the second day after infection, cell lysates were prepared and subjected to immunoprecipitation (IP) with polyclonal anti-HBV core protein antibody. Samples were then resolved by SDS-13% PAGE and analyzed by immunoblotting with anti-VSV antibody (upper panel) or anti-HBV core protein antibody (middle panel). In parallel, total cell lysates were analyzed by immunoblotting with anti-VSV antibody (lower panel). Positions of SRPKs and HBV core protein are indicated on the right. (B) HuH-7 cells were transiently transfected in 10-cm dishes with control plasmid, replication-competent HBV plasmid pSPT1.2xHBV (15 μg/dish), or plasmid encoding SRPK2-VSV (5 μg/dish), as indicated. After 48 h, cell lysates were prepared and processed as described for panel A. In addition, total cell lysates were analyzed by immunoblotting with anti-HBsAg antibody (lower panel). Positions of SRPK2, HBV core protein, and the different forms of HBsAg are indicated on the right.

Comparison of binding characteristics of HBV core kinase activity and SRPKs for different chromatography media. Total cellular extracts of HuH-7 cells were adjusted to different NaCl concentrations and then either control incubated without chromatography media orincubated in the presence of Q Sepharose (A), SP Sepharose (B), heparin-Sepharose (C), or phosphocellulose beads (D). Supernatants were then assayed for HBV core kinase activity in an in vitro kinase assay with GST-HBV core protein C1 as a substrate. After gel electrophoresis, phosphate incorporation into substrate protein was visualized by autoradiography (upper panels). In parallel, the same supernatants were resolved on GST-HBV-C1-containing SDS gels and subjected to an in-gel kinase assay. Phosphorylation of copolymerized GST-HBV-C1 by renatured cellular kinases was detected by autoradiography (second panels from the top). In addition, identical samples were subjected to immunoblotting with either anti-SRPK1 or anti-SRPK2 antibodies (lower two panels). Positions of GST-HBV-C1, SRPK1, and SRPK2 are indicated on the right. The position of an unidentified 42-kDa kinase detected in the in-gel kinase assay is marked by an arrow.

Analysis of in vivo phosphorylation and SRPK-mediated in vitro phosphorylation of HBV core protein. (A) HuH-7 cells were transiently transfected in six-well dishes with 1.5 μg of either empty expression vector, plasmid encoding wild-type HBV core protein, or plasmid encoding mutant HBV core protein devoid of the three reported C-terminal serine phosphorylation sites (serines 155, 162, and 170) per well. On the second day after transfection, cells were labeled with 50 μCi of ³³P_i per ml for 3.5 h. Lysates were then subjected to immunoprecipitation (IP) with anti-HBV core antibody. After gel electrophoresis and transfer onto a nitrocellulose membrane, HBV core protein phosphorylation was first visualized by autoradiography (upper panel), followed by immunoblotting of the same filter with anti-HBV core protein antibody (lower panel). (B) COS-7 cells were transiently transfected in six-well dishes with either control plasmids or expression constructs encoding FLAG-tagged SRPK1 or VSV-tagged SRPK2 (1.5 μg/well each). After cell lysis and immunoprecipitation (IP) with anti-FLAG or anti-VSV antibodies, in vitro kinase reactions were performed in the presence of [γ-³²P]ATP and GST-HBV-C1 core fusion protein with (C1) or without the three previously mapped C-terminal serine phosphorylation sites (C1-AAA, serines 155, 162, and 170 mutated to alanines). After gel electrophoresis, phosphate incorporation into substrate protein was visualized by autoradiography. (C) HuH-7 cells were transiently transfected in six-well dishes with plasmids encoding either wild-type HBV core protein or its mutants lacking serine phosphorylation sites in the C-terminal part as indicated (1.5 μg/well each). After metabolic labeling with 50 μCi of ³³P_i per ml for 3.5 h, cell lysates were processed as described for panel A. HBV core protein phosphorylation was visualized by autoradiography (upper panel) and quantified by a PhosphorImager, and the same filter was then probed with anti-HBV core protein antibody (lower panel).