FIG 7.

Rescue of expression and assembly of CTD-deleted HBc by WT HBc in HEK293 cells. (A) Cytoplasmic lysate (20 μl) from HEK293 cells transiently transfected with the indicated plasmids was resolved by SDS-PAGE and detected by Western blot analysis using the anti-HBc NTD MAb. The control plasmid expresses the neomycin resistance gene and was used as a filler. The ratios (9:1 and 1:9) refer to the amount of the first plasmid (always expressing an HBc protein) relative to the amount of the second plasmid (expressing either no HBc or the indicated HBc protein). (B) Capsids in cytoplasmic lysate of HEK293 cells cotransfected with both the WT HBc- and C149-expressing plasmids were fractionated on a linear 15% to 30% sucrose gradient in an SW32 rotor at 27,000 rpm for 4 h at 4°C (the direction of centrifugation is indicated by the arrow). The indicated fractions (lanes 2 to 13), along with the input lysate (lane 1), were resolved by SDS-PAGE, and WT HBc and C149 were detected by Western blot analysis using the anti-HBc NTD MAb. (C) Coimmunoprecipitation of C149 and WT HBc in mosaic capsids. The capsid peak fraction (fraction 9) from the sucrose gradient was subjected to immunoprecipitation using the anti-CTD MAb (6-1) (lane 2) or a control IgG (lane 1). The immunoprecipitates were detected by the anti-NTD MAb following SDS-PAGE and Western blot analysis. (D) Cytoplasmic lysate from HEK293 cells transiently transfected with WT (lanes 1 and 3) or C182 (lanes 2 and 4) was resolved by SDS-PAGE and detected by Western blot analysis using the anti-NTD MAb (lanes 1 and 2) or anti-CTD MAb 6-1 (lanes 3 and 4). C, WT HBc monomer; C149 monomer, C-terminally truncated HBc protein (terminated at position 149); IP, immunoprecipitation. C182 comigrated with WT HBc.