Journal: Microbiology Spectrum

Article Title: Hepatitis B Virus X Protein Stimulates Hepatitis C Virus (HCV) Replication by Protecting HCV Core Protein from E6AP-Mediated Proteasomal Degradation

doi: 10.1128/spectrum.01432-22

Figure Lengend Snippet: HBx stabilizes HCV core protein by protecting it from ubiquitin-dependent proteasomal degradation. (A) HepG2 cells were transfected with HA-Core with or without HA-HBx for 48 h, treated with 50 μM cycloheximide (CHX) for the indicated times, and collected for Western blotting. The band intensity of HA-Core was quantified as described in the legend of Fig. 1A to determine the half-life ( t 1/2 ) of the HCV core protein ( n = 4). (B) Huh7D cells were transfected with the 1.2-mer WT replicon and then infected with HCV at an MOI of 10 for an additional 48 h. The t 1/2 value of the HCV core protein was determined as described above for panel A. (C) Huh7D-NTCP cells were coinfected with HBV at an MOI of 30 and HCV at an MOI of 10 for 48 h. The t 1/2 value of the HCV core protein was determined as described above for panel A. (D) HepG2 cells were transfected with HA-HBx, HA-Core, an E6AP expression plasmid, and HA-ubiquitin (HA-Ub) for 48 h. Total HA-Core proteins were immunoprecipitated with an anti-HCV core protein antibody and subjected to Western blotting using an anti-HA antibody to detect polyubiquitinated HA-Core. The input shows the levels of the indicated proteins in whole-cell lysates. (E) HepG2 cells were transfected with the indicated amounts of HA-HBx, HA-Core, scrambled (SC) shRNA, and E6AP shRNA for 48 h, and samples were collected for immunoprecipitation (IP), as described above for panel D. (F) Huh7D-NTCP cells were transfected with an empty vector, an E6AP expression plasmid, and HA-Ub for 24 h and then monoinfected with HCV or coinfected with HBV and HCV for 48 h as described above for panel C, and samples were collected for IP as described above for panel D. (G) Huh7D cells were transfected with an empty vector, the 1.2-mer WT replicon, an E6AP expression plasmid, and HA-Ub for 24 h and then infected with HCV at an MOI of 10 for an additional 48 h. (H) HepG2 cells were transfected with either HA-HBx or HA-Core, followed by Western blot analysis. (I) HepG2 cells were transfected with the indicated amounts of HA-HBx, HA-Core, and an E6AP expression plasmid for 48 h. (J) Huh7D cells were transfected with the 1.2-mer WT replicon and an E6AP expression plasmid for 24 h and then infected with HCV at an MOI of 10 for an additional 48 h. (K) Huh7D-NTCP cells were transfected with either an empty vector or an E6AP expression plasmid for 24 h and then either mock infected or infected with HBV at an MOI of 30 and/or HCV at an MOI of 10 for an additional 48 h. (L) HepG2 cells were transfected with the indicated amounts of HA-HBx and HA-Core for 44 h and then either mock treated or treated with 10 μM MG132 for an additional 4 h. (M) HepG2 cells were transfected with the 1.2-mer WT replicon and then infected with HCV as described above for panel J. For lanes 4 to 6, cells were either mock treated or treated with 10 μM MG132 for 4 h before harvest.

Article Snippet: The E6AP expression plasmid pCMVT N-HA-hE6AP (catalog no. 37601), encoding full-length human HA-tagged E6AP, was purchased from Addgene.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Infection, Expressing, Plasmid Preparation, Immunoprecipitation, shRNA

Journal: Microbiology Spectrum

Article Title: Hepatitis B Virus X Protein Stimulates Hepatitis C Virus (HCV) Replication by Protecting HCV Core Protein from E6AP-Mediated Proteasomal Degradation

doi: 10.1128/spectrum.01432-22

Figure Lengend Snippet: HBx upregulates HCV core protein levels by repressing E6AP expression via DNA methylation. (A) HepG2 cells were transfected with the indicated amounts of HA-Core and HA-HBx for 48 h. DNA methyltransferase (DNMT) activity in the cell extracts was determined ( n = 6). The levels of the indicated proteins were determined by Western blotting. (B) Methylation-specific PCR (MSP) was performed to determine whether the CpG sites within the E6AP promoter were unmethylated (U) or methylated (M) in cells prepared as described above for panel A. (C) Huh7D cells were transfected with the 1.2-mer WT replicon for 24 h and then infected with HCV at an MOI of 10 for an additional 48 h. DNMT activity in the cell extracts was determined ( n = 4). The levels of the indicated proteins were determined by Western blotting. (D) MSP was performed as described above for panel B with cells prepared as described above for panel C. (E) Huh7D-NTCP cells were either mock infected or infected with HBV at an MOI of 30 and/or HCV at an MOI of 10 for 48 h. The DNMT activity in the cell extracts was determined ( n = 4). (F) MSP was performed as described above for panel B with cells prepared as described above for panel E. (G) HepG2 cells were transfected with the indicated amounts of HA-Core and HA-HBx for 48 h. For lanes 4 to 6, cells were treated with 10 μM 5-aza-2′dC for 24 h before harvest. (H) HepG2 cells were transfected with the indicated amounts of HA-Core and HA-HBx in the presence and absence of the DNMT1 shRNA plasmid for 48 h. OD 450 , optical density at 450 nm.

Article Snippet: The E6AP expression plasmid pCMVT N-HA-hE6AP (catalog no. 37601), encoding full-length human HA-tagged E6AP, was purchased from Addgene.

Techniques: Expressing, DNA Methylation Assay, Transfection, Activity Assay, Western Blot, Methylation, Infection, shRNA, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: Hepatitis B Virus X Protein Stimulates Hepatitis C Virus (HCV) Replication by Protecting HCV Core Protein from E6AP-Mediated Proteasomal Degradation

doi: 10.1128/spectrum.01432-22

Figure Lengend Snippet: HBx stimulates HCV replication by repressing E6AP expression via DNA methylation. (A) Huh7D cells were transfected with HA-HBx and an E6AP expression plasmid for 24 h and then infected with HCV at an MOI of 10 for an additional 48 h, followed by Western blotting. (B) Levels of extracellular HCV particles from Huh7D cells prepared as described above for panel A were measured by qRT-PCR as described in the legend of Fig. 1B ( n = 4). (C) Huh7D cells were transfected with HA-HBx and then infected with HCV as described above for panel A, in the presence or absence of 10 μM 5-aza-2′dC. (D) Levels of extracellular HCV RNA from Huh7D cells prepared as described above for panel C were measured as described in the legend of Fig. 1B ( n = 5). (E) Huh7D cells were transfected with HA-HBx and a DNMT1 shRNA plasmid and then infected with HCV as described above for panel A, followed by Western blotting. (F) Levels of extracellular HCV RNA from Huh7D cells prepared as described above for panel E were measured as described in the legend of Fig. 1B ( n = 5). (G) Huh7D cells were transfected with the 1.2-mer WT replicon and an E6AP expression plasmid and then infected with HCV as described above for panel A. (H) Levels of extracellular HCV RNA from cells prepared as described above for panel G were measured as described in the legend of Fig. 1B ( n = 3). (I) Huh7D cells were transfected with the 1.2-mer WT replicon and then infected with HCV as described above for panel A. For lanes 4 and 5, cells were treated with 10 μM 5-aza-2′dC, as described in the legend of Fig. 4G . (J) Levels of extracellular HCV RNA from cells prepared as described above for panel I were measured as described in the legend of Fig. 1B ( n = 5). (K) Huh7D cells were transfected with the 1.2-mer WT replicon and a DNMT shRNA plasmid and then infected with HCV as described above for panel A. (L) Levels of extracellular HCV RNA from cells prepared as described above for panel K were measured as described in the legend of Fig. 1B ( n = 5). (M) Huh7D cells were transfected with HA-HBx and an E6AP shRNA plasmid and then infected with HCV as described above for panel A, followed by Western blotting. (N) Levels of extracellular HCV RNA from Huh7D cells prepared as described above for panel M were measured as described in the legend of Fig. 1B ( n = 5). (O) Huh7D cells were transfected with the 1.2-mer WT replicon and E6AP shRNA and then infected with HCV as described above for panel A. (P) Levels of extracellular HCV RNA from cells prepared as described above for panel O were measured as described in the legend of Fig. 1B ( n = 5).

Article Snippet: The E6AP expression plasmid pCMVT N-HA-hE6AP (catalog no. 37601), encoding full-length human HA-tagged E6AP, was purchased from Addgene.

Techniques: Expressing, DNA Methylation Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Quantitative RT-PCR, shRNA

Journal:

Article Title: 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

doi: 10.1016/j.bbrc.2010.05.092

Figure Lengend Snippet: A. Plasmid expression vectors for Flag-tagged 55K and 42K CDK9 proteins were transfected into HeLa cells and immunoprecipitations with the Flag antibody were performed at Day 2 post-transfection. An immunoblot was performed to measure the expression levels of the Flag-tagged proteins and the relative amount of Cyclin T1 that co-immunoprecipitated with each protein. B. The plasmid vectors were transfected into HeLa cells and immunoprecipitations with the Flag antibody were performed. A portion of the immunoprecipitate was analyzed on a SDS-polyacrylamide gel which was stained with Colloidal Blue. The remaining portion was subjected to a proteomics analysis to identify proteins present in the precipitations.

Article Snippet: Cyclin T1, Cyclin T2a, CDK7 and CDK9 antisera were purchased from Santa Cruz Biotechnology; γ-H2AX antiserum was from Millipore; β-actin and Flag antisera were from Sigma; PARP antiserum was from Cell Signaling; anti-Flag M2 affinity gel was from Sigma; Ku70 antisera was from Abcam; mouse anti-rabbit light-chain antiserum was from Jackson Immunoresearch.

Techniques: Plasmid Preparation, Expressing, Transfection, Western Blot, Immunoprecipitation, Staining