Calcium Signaling by HBx Protein in Hepatitis B Virus DNA Replication Michael J. Bouchard, Li-Hua Wang, and Robert J. Schneider

Supplementary Material

Plasmids have all been described previously. Plasmid expressing HBx (pAdCMVX), control vectors pAdCMV, pAdCMVXo, or BS, and the AP1 promoter driven luciferase reporter plasmid were described in (1, 2). The CREB driven luciferase reporter plasmid was described in (3). The Pyk2 kinase mutant pPKM and the vector control pRK5 were described in (4). Replication-competent HBV genomic DNA plasmids pHBV (wild type) and pHBV(-HBx) mutant was described in (5).Transient transfections were performed with Lipofectin (Life Technologies) for Chang and HepG2 cells, according to the manufacturer's directions. 10⁷ cells were transfected overnight, allowed to recover for 12 h and then serum starved for 16 h prior to analysis for luciferase activity. Transfections used 6 g of pPKM plasmid or pRK5 empty plasmid, 0.4 to 3 g of pAdCMVX, pAdCMVXo (which expresses an HBx mRNA devoid of AUG initiation codons) or empty plasmid pAdCMV or BS (1, 2), and/or 2 g of pHBV wild type or pHBV(-HBx) genomic DNA plasmids (5). Analysis of HBV replication in cytoplasmic core particles was performed 4 days post-transfection as described (6).

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Supplemental Figure 1. Chang or HepG2 cells were transfected with pBS empty plasmid (vector) or pAdCMVX (HBx) expression plasmid, 2 g of luciferase reporter plasmid controlled by a minimal TATA-box promoter and 4 copies of an AP-1 binding site and pPKM plasmid or pRK5 empty plasmid. (A) Equal protein amounts were assayed for luciferase activity. For analysis of low level expression of HBx, 0.4 g (1/8) of pAdCMVX was transfected. A typical experiment is shown. (B) Chang cell lysates were prepared in a modified RIPA buffer (1), gel-electrophoresis and immunoblot analysis was performed with anti-Pyk2 or anti-Pyk phosphotyrosine-402 (Y(P)-402) antibodies (Biosource, Int.). Nontransfected cells were treated with 20 ng/ml phorbol ester TPA for 20 min to activate Pyk2 (TPA samples). (C) Equal amounts of protein lysates were immunoprecipitated with antibodies to Pyk2, an in vitro kinase assay was performed using [-³²P]ATP as described (6), and phosphorylation of associated Src, Fyn, and Pyk2 analyzed by gel-electrophoresis and autoradiography. Identification of Pyk2 and Src-Fyn proteins, which electrophoretically comigrated, was performed by immunoblot with specific antisera.

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Supplemental Figure 2. Chang cells were transfected with expression vectors pAdCMVX (HBx), pAdCMVXo, pPKM, or empty vectors pAdCMV or pRK5. (A) Chang cells were treated with 50 M BAPTA-AM for 2 h, equal protein amounts resolved by gel-electrophoresis and immunoblotted with antibodies to Pyk2 or activated Y-402 phosphorylated Pyk2. TPA treatment was for 20 min using 20 ng/ml. (B) Chang cells transfected with vector or pAdCMVX were treated 2h with 0.5 mM or 3 mM EGTA, and Pyk2 examined by immunoblot.

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