Journal: PLoS Pathogens

Article Title: Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

doi: 10.1371/journal.ppat.1010057

Figure Lengend Snippet: (A) Illustration of wild-type precore protein (p25-WT) and mutant/HA-tagged p25 as well as its proteolytically processed products p22 and p17 that are expressed by pXF3H-derived plasmids (see for detailed description). Red arrow indicates the mutation of Cp starting codon (AUG) to AUA. The gray box indicates the insertion of HA tag at the immediate upstream of Cp starting codon. (B) HepG2 cells were transfected with pXF3H-p25-WT and derived plasmid expressing p25 with the indicated single amino acid substitution. The cells were harvested at 48 h post transfection. Intracellular p25-derived proteins were detected by Western blot assay with an antibody recognizing the last 14 amino acid residues at the C-terminus of precore protein and Cp (anti-HBc-170A). HBeAg in culture media was detected by CLIA and results (mean ± SD) from three independent experiments were analyzed by two-tailed Student’s t-test (unpaired), ***: P < 0.001. (C) HepG2 cells were transfected with a pXF3H-derived plasmid expressing the indicated p25 proteins or pCMV-HBc expressing Cp. Intracellular p25-derived proteins or Cp were detected by Western blot assay with an anti-HBc-170A as the primary antibody. The lysate from AML12HBVpolY63F cells was analyzed in parallel to serve as the control of hyper- and hypo-phosphorylated Cp. (D) HepG2 cells were transfected with pXF3H-p25HA, harvested at 48 h post transfection and lysed with core DNA lysis buffer. Aliquots of the cell lysate were mock-treated or treated with Lambda phosphatase at 30°C for 40 min. HBV p22 in the reactions were detected by Western blot assay with anti-HBc-170A. ( E ) HepG2 cells were transfected with a plasmid expressing the indicated precore protein and harvested at 48 h post transfection. The cells were lysed by 1× LDS lysis buffer (contained 2.5% B-ME) and the lysates were resolved by electrophoresis in NuPAGE 12% Bis-Tris Protein Gel (upper panel) and 12% phos-tag gel (lower panel). After blotting onto membranes, p22 were detected by anti-HBc-170A as primary antibody. β-actin served as a loading control.

Article Snippet: HepG2 cells transfected with desired plasmids were lysed by core DNA lysis buffer, 40 μl lysed samples were incubated with 1μl Lambda phosphatase (NEB, MA, USA), 5 μl 10 ×NEB buffer 3.1 and 4 μl nuclease-free water for 40 min at 30°C.

Techniques: Mutagenesis, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Two Tailed Test, Lysis, Electrophoresis

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: P-Cell separation of replication proteins from Ad-infected cells. (A) Western blots of fractions I, IIA, IIB, and IIC with Pol δ, Pol ɛ, Pol α, Ad DBP, RFC, RPA, and PCNA antibodies. (B) Reconstitution of AAV DNA replication in vitro using fractionated Ad-infected-cell extracts. Standard replication reaction mixtures (30 μl) contained either crude Ad-infected-cell extract (C; 200 μg) or P-Cell fractions (I, 160 μg; IIA, 36 μg; IIC′, 30 μg; IID, 9 μg) and Rep78 (3.4 μg), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. The DNA products from each reaction were digested with DpnI and analyzed on 0.8% Tris-borate-EDTA (TBE)-agarose. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion. DNA products sensitive to DpnI digestion are denoted by the black line. (C) Optimization of AAV DNA replication with RFC and P-Cell fractions I and IIA. Standard replication reaction mixtures (15 μl) contained either crude Ad-infected-cell extract (C; 100 μg) or P-Cell fraction I (80 μg), RFC (0.5 μg), Rep78 (1.7 μg), and various amounts of fraction IIA (IIA total protein concentration, 6 mg/ml), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. Half of each reaction mixture was subjected to DpnI digestion and analyzed on 0.8% TBE-agarose; the remaining half was used to quantify [32P]dAMP incorporation using a DE-81 filter binding assay (expressed as pmol of dAMP incorporated per 15 μl reaction mixture). The migration pattern and fragment size of radiolabeled HindIII-digested lambda DNA are indicated on the left.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot, In Vitro, Incubation, Protein Concentration, Filter-binding Assay, Migration, Lambda DNA Preparation

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: (A) Silver-stained polyacrylamide gel of fractions used in this study and purified from Ad-infected-cell extracts. Lane C, Ad-infected-cell extract (2 μl, 92 μg); IIA, 5 μl, 55 μg; IIB, 5 μl, 30 μg; IIC, 5 μl, 25 μg; I, 5 μl, 115 μg; IA, 5 μl, 34 μg; IB, 5 μl, 40 μg; IC, 5 μl, 4.7 μg. (B) Silver stain of an SDS-polyacrylamide gel of purified protein fractions: DEAE Pol δ from uninfected extract (15 μl, 16.5 μg), Ad BDP (5 μl, 0.65 μg), PCNA (2.5 μl, 0.7 μg), baculovirus-expressed RPA (20 μl, 5 μg), RFC from Ad-infected-cell extract (40 μl, 0.6 μg), Pol ɛ from uninfected extract (40 μl, 3.8 μg), and baculovirus-expressed Pol δ (30 μl, 3.2 μg). Arrows indicate the molecular masses of the expected subunits in each complex.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Staining, Purification, Infection, Silver Staining

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: Q-Sepharose chromatography of P-Cell fraction I from Ad-infected cells. (A) Western blot analysis of Q-Sepharose fractions IA, IB, IC, and ID. RPA (lane 1, 0.7 μg), PCNA (lane 2, 1.7 μg), P-Cell fraction I (400 μg), and Q-Sepharose fractions IA (250 μg), IB (188 μg), IC (60 μg), and ID (6.3 μg) were resolved on 10% SDS-polyacrylamide and then transferred to nitrocellulose. The nitrocellulose filter was probed with RPA (70-kDa subunit) and PCNA monoclonal antibodies. (B) Reconstitution of AAV DNA replication with Q-Sepharose fractions. Standard replication reaction mixtures (15 μl) contained P-Cell fractions I (80 μg) and IIA (24 μg); RFC (0.5 μg); Rep78 (1.7 μg); and Q-Sepharose fractions IA (50 μg), IB (38 μg), IC (12 μg), and ID (1.3 μg), as indicated. Reaction mixtures were incubated for 2 h at 37°C and processed as described in Materials and Methods. md and dd indicate monomer duplex and dimer duplex DNA species, respectively. (C) Replacement of Q-Sepharose fraction IC with PCNA. Standard replication reaction mixtures (15 μl) contained P-Cell fraction IIA (24 μg), RFC (0.5 μg), Rep78 (1.7 μg), Q-Sepharose fractions IA (50 μg) and IC (12 μg), RPA (0.7 μg), and PCNA (lanes 6 to 10, 17 ng, 34 ng, 85 ng, 170 ng, and 340 ng, respectively; lanes 11 and 12; 340 ng), as indicated. Reaction mixtures were incubated for 2 h at 37°C and processed as described in Materials and Methods.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Chromatography, Infection, Western Blot, Bioprocessing, Incubation

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: Q-Sepharose chromatography of P-Cell fraction IIA from Ad-infected cells. (A) DNA polymerase activity profile of Q-Sepharose fractions. DNA polymerase activity of each fraction was assayed by measuring the incorporation of dAMP into activated DNA. Standard reaction mixtures (25 μl) contained 3 μl of each fraction and were incubated for 30 min at 37°C. Incorporation of dAMP into DNA was quantified using a DE-81 filter binding assay and is expressed as dAMP (pmol) incorporated per 25 μl reaction mixture. IIA1, IIA2, and IIA3 represent pooled fractions used to reconstitute DNA replication. (B) PCNA-dependent and independent DNA polymerase activities of Q-Sepharose fraction IIA-2 as described in Materials and Methods (PCNA, 255 ng). (C) PCNA-dependent and independent DNA polymerase activities of Q-Sepharose fraction IIA-3 as described in Materials and Methods (PCNA, 255 ng). (D) Reconstitution of AAV DNA replication with Q-Sepharose fractions. Standard replication reaction mixtures (15 μl) contained P-Cell fraction I (80 μg); RFC (0.5 μg); Rep78 (1.7 μg); IIA (24 μg); or Q-Sepharose fractions IIA1 (5 μg), IIA2 (10 μg), and IIA3 (7.5 μg), as indicated. md and dd indicate monomer duplex and dimer duplex DNA species, respectively. (E) Titration of Q-Sepharose fraction IIA2. Standard replication reaction mixtures (15 μl) were as described above except that various amounts of IIA2 (lanes 5 to 8, 3 μg, 6 μg, 12 μg, and 18 μg, respectively) were used to substitute for fraction IIA. All reaction mixtures were incubated for 4 h at 37°C and processed as described in Materials and Methods. [32P]dAMP incorporation was measured as described for Fig. ​Fig.33.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Chromatography, Infection, Activity Assay, Incubation, Filter-binding Assay, Titration

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: DEAE-Sepharose fractionation of DNA polymerases from P-Cell fraction IIA of uninfected-293 cell extract. (A) DNA polymerase activity of fractions from DEAE Sepharose (see Materials and Methods). (B) Western blot of DEAE fractions using anti-Pol δ and anti-Pol ɛ antibodies which were probed for sequentially without stripping the blot between antibodies. (C) In vitro AAV DNA replication. Standard replication reaction mixtures (15 μl) contained, as shown, the following components: crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), DEAE-Sepharose fractions 10 and 11 of the Pol δ pool (0.44 mg/ml), and fractions 13 to 15 of the Pol ɛ pool (2.6 mg/ml), as indicated. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Fractionation, Activity Assay, Western Blot, Stripping Membranes, In Vitro

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: Comparison of DNA Pol δ and Pol ɛ. (A) Western blot using anti-Pol δ and anti-Pol ɛ (blot probed sequentially with antibodies) shows that fractions of the DEAE fraction of Pol δ (shown in Fig. ​Fig.7)7) did not contain Pol ɛ and that the Mono S fraction of Pol ɛ did not contain Pol δ. (B) In vitro AAV DNA replication with various purified DNA replication components. Standard replication reaction mixtures (15 μl) contained crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), DEAE Pol δ (2 μg, 16 nmol [3H]dTTP/h/μl), and Mono S Pol ɛ (0.4 μg, 348 nmol [3H]dTTP/h/μl), as indicated. md and dd indicate monomer duplex and dimer duplex DNA species, respectively.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Comparison, Western Blot, In Vitro, Purification

Journal:

Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

doi: 10.1128/JVI.02651-06

Figure Lengend Snippet: Replacement of Pol δ purified from 293 cell extracts with baculovirus-expressed Pol δ in the in vitro DNA replication assay. Standard replication reaction mixtures (15 μl) contained crude RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), DEAE Pol δ (0.43 mg/ml, 7 nmol [3H]dTTP/h/μl), baculovirus-expressed Pol δ (0.024 mg/ml, 1.7 nmol [3H]dTTP/h/μl), RPA (30 to 260 ng), and Ad DBP (15 to 130 ng), as indicated. The amount of 32P incorporation for the monomer AAV replication species was determined by phosphorimager analysis and is indicated for each lane. md and dd indicate monomer duplex and dimer duplex DNA species, respectively.

Article Snippet: Mouse monoclonal PCNA antibody (PC10) was purchased from Santa Cruz Biotechnology.

Techniques: Purification, In Vitro