Journal: Journal of Virology

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

doi: 10.1128/JVI.00539-17

Figure Lengend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

Article Snippet: For Exo I and Exo III (Exo I & III) digestion, 20 μl PF DNA prepared as described above was treated with 0.25 μl each of Exo I (NEB; 5 units) and Exo III (NEB; 25 units) in 1× NEB Cutsmart buffer (50 mM potassium acetate, 20 mM Tris–acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin [BSA], pH 7.9 [prepared at 25°C]) or 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 [prepared at 25°C]), as indicated, at 37°C for 2 to 3 h in a total volume of 23 μl.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

Journal: Journal of Virology

Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

doi: 10.1128/JVI.00539-17

Figure Lengend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

Article Snippet: For Exo I and Exo III (Exo I & III) digestion, 20 μl PF DNA prepared as described above was treated with 0.25 μl each of Exo I (NEB; 5 units) and Exo III (NEB; 25 units) in 1× NEB Cutsmart buffer (50 mM potassium acetate, 20 mM Tris–acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin [BSA], pH 7.9 [prepared at 25°C]) or 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 [prepared at 25°C]), as indicated, at 37°C for 2 to 3 h in a total volume of 23 μl.

Techniques: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker

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

doi: 10.1128/JVI.00394-16

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

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

doi: 10.1128/JVI.00394-16

Figure Lengend Snippet: Effects of exogenous phosphatase and RNase treatment on capsid assembly in RRL. The indicated HBc proteins were translated in RRL, and the translation reaction mixtures were resolved by agarose gel electrophoresis (top panels) or SDS-PAGE (bottom panels) without any further treatment (lanes 1, 7, 13, 19, 25, and 31) or were treated with NEB buffer 3 alone overnight at 37°C (buffer) (lanes 2, 8, 14, 20, 26, and 32), with buffer 3 plus CIAP overnight at 37°C (CIAP) (lanes 3, 9, 15, 21, 27, and 33), with buffer 3 plus CIAP overnight at 37°C followed by RNase treatment for one additional hour (CIAP-RNase) (lanes 4, 10, 16, 22, 28, and 34), with RNase for 1 h followed by buffer 3 plus CIAP overnight at 37°C (lanes 5, 11, 17, 23, 29, and 35), or with the mixture of phosphatase inhibitors overnight at 37°C (lanes 6, 12, 18, 24, 30, and 36). All lanes contained 2 μl translation products. 35 S-labeled HBc proteins were detected by autoradiography. C, 3A, and 3E/7A, WT or mutant HBc subunits; C149, C-terminally truncated HBc protein (terminated at position 149); C-deP, dephosphorylated 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: Agarose Gel Electrophoresis, SDS Page, Labeling, Autoradiography, Mutagenesis