Journal: Journal of Virology
Article Title: Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation
Figure Lengend Snippet: HBV capsid assembly in RRL and effects of exogenous phosphatase and phosphatase inhibitors on assembly. The WT and mutant HBc proteins or the control luciferase (Luc) was translated in RRL. All samples were resolved by agarose gel electrophoresis. (A) The indicated protein translated in RRL was incubated overnight at 37°C in 1× NEB restriction digestion buffer 3 alone (lanes 1, 3, 5, 7, and 9) or with CIAP (lanes 2, 4, 6, 8, and 10) before resolution on the gel. The recombinant HBV capsid (rHBc) purified from E. coli was loaded in lane 11. (B) The indicated translation reaction mixture was loaded directly following translation upon dilution in double-distilled water (dH 2 O) and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, 9, and 13), after dilution in NEB buffer 3 (buffer 3) but without the overnight incubation (lanes 2, 6, 10, and 14), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, 11, and 15), or after overnight incubation in buffer 3 and with CIAP (lanes 4, 8, 12, and 16). (C) The indicated translation reaction mixture was loaded directly following translation upon dilution in dH 2 O and without the overnight incubation (i.e., no assembly reaction) (lanes 1, 5, and 9), after dilution in dH 2 O and with incubation overnight at 37°C (lanes 2, 6, and 10), after dilution in buffer 3 and with incubation overnight at 37°C (lanes 3, 7, and 11), or after overnight incubation at 37°C in buffer 3 and with a mixture of phosphatase inhibitors (PPI) (lanes 4, 8, and 12). Each lane contained 3 μl translation product except that 3.125 ng rHBc was loaded in lane 11 of panel A. 35 S signals were detected by autoradiography (top). The HBc proteins were also detected by the MAb antibody against the NTD (bottom). C/3A/3E, WT, 3A, or 3E HBc subunits (i.e., not present in the capsid); C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated WT HBc subunits; Ca, capsids.
Article Snippet: Unless specifically indicated otherwise, the general assembly reaction mixtures included 1 to 3 μl of translation products per 10 μl final reaction volume in 1× buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9; New England BioLabs, or NEB) supplemented with 1× EDTA-free protease inhibitor cocktail (Roche) and 0.8 U/μl RNasin Plus RNase inhibitor (Promega).
Techniques: Mutagenesis, Luciferase, Agarose Gel Electrophoresis, Incubation, Recombinant, Purification, Autoradiography