FIG.5.

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).