Research ArticleVIROLOGY

Restriction of hepatitis B virus replication by c-Abl–induced proteasomal degradation of the viral polymerase

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Science Advances  06 Feb 2019:
Vol. 5, no. 2, eaau7130
DOI: 10.1126/sciadv.aau7130
  • Fig. 1 c-Abl kinase reduces HBV replication and HBV polymerase protein level.

    (A and B) Quantitation of capsid-associated viral DNA by real-time polymerase chain reaction (PCR). HepG2.2.15 cells were treated with dimethyl sulfoxide (DMSO) or imatinib (A) or dasatinib (B) in indicated concentrations for 24 hours before harvest. Mean copy number from cells treated with DMSO was set to 100% and compared with others. Statistical significance compared with DMSO is noted by asterisks (n = 3 to 4 per group). (C) Quantitation of capsid-associated viral DNA by real-time PCR in HepG2.2.15 cells knocking out control (sgCtrl) or Abl (sgAbl-1/2). Mean copy number from sgCtrl cells was set to 100% and compared with others (n = 3 per group). (D and E) Quantitation of capsid-associated viral DNA by real-time PCR in HepG2 cells (D) or Huh7 cells (E) knocking out control or Abl. Cells were transfected with pHBV for 48 hours before harvest. Mean copy number from sgCtrl cells was set to 100% and compared with others (n = 3 per group). (F) Human embryonic kidney (HEK) 293T cells were cotransfected with constructs expressing hemagglutinin (HA)–tagged polymerase (HA-Pol), preS (HA-preS), preC (HA-preC), and HBx (HA-HBx), and Flag-tagged Abl (Flag-Abl) or empty vector controls. SE, short exposure; LE, long exposure. Western blot was performed 48 hours after transfection. HepG2 cells (G) or Huh7 cells (H) were transfected as shown. Cells were treated with DMSO or 2 μM imatinib for 24 hours before harvest. Total cell lysates were then analyzed for the indicated proteins. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 c-Abl kinase promotes HBV polymerase ubiquitination and proteasome-dependent degradation.

    (A) CHX chasing analysis for polymerase stability in HEK293T cells expressing Flag-Abl. Cells were treated with CHX (100 μg/ml) for the indicated time. (B) Time course of polymerase protein decay from (A). Each band of the Western blot for polymerase was quantified with National Institutes of Health (NIH) ImageJ software. Half-life (t1/2) was estimated as the time for degradation of 50% of the protein. (C) HEK293T cells were transfected with HA polymerase, Flag-Abl, or empty vector control and were treated with MG132 (20 μM) or MLN4924 (0.2 μM) for 8 hours before harvest. Total cell lysates were subjected to Western blotting.

  • Fig. 3 CRL4cdt2 E3 ubiquitin ligase targets HBV polymerase for ubiquitination.

    (A) Co-IP of HA polymerase with CRL4 components in HEK293T cells by overexpression HA polymerase, Myc-CUL4A, Myc-CUL4B, and Flag-DDB1. (B) Co-IP of HA polymerase with endogenous CRL4 in HEK293T cells. (C) Ubiquitination of polymerase by Flag IP in Huh7 cells expressing HA polymerase, Flag-ub, and siDDB1, siCUL4A, siCUL4B, or a combination of siCUL4A and siCUL4B and treated with MG132 for 8 hours before harvest. Flag immunoprecipitates (top) or total cell lysates (bottom) were then analyzed by Western blot. (D) Immunoblot for polymerase protein level in Huh7 cells knocking down CRL4 components. (E) Co-IP of HA polymerase with endogenous Cdt2 and DDB1 in Huh7 cells. (F) The transfected HEK293T cells were treated with DMSO or MLN4924 (0.2 μM) for 8 hours before harvest. (G) CHX chasing analysis for polymerase stability in HEK293T cells expressing Flag-Cdt2. Cells were treated with CHX (100 μg/ml) for the indicated time. (H) Time course of polymerase protein decay from Fig. 3G. Each band of the Western blot for polymerase was quantified with NIH ImageJ software. Half-life (t1/2) was estimated as the time for degradation of 50% of the protein. (I) HEK293T cells were transfected with control siRNA or siCdt2 and HA polymerase. Total cell lysates were subjected to Western blotting. (J) Co-IP of DDB1 with polymerase in Huh7 cells by overexpressing HA polymerase, Myc-DDB1, and siCdt2

  • Fig. 4 c-Abl kinase activates CRL4-DDA1 to reduce the level of HBV polymerase.

    (A) Immunoblot for polymerase protein level in HEK293T cells overexpressing Flag-Abl and knocking down DDB1 or CUL4A/4B. (B) Immunoblot for polymerase protein level in HEK293T cells overexpressing Flag-Abl and knocking down Cdt2. (C) Ubiquitination of polymerase by IP in Huh7 cells expressing HA polymerase, Flag-Abl, Myc-ub, and siDDB1, siCUL4A, siCUL4B, or a combination of siCUL4A and siCUL4B and treated with MG132 for 8 hours. IP-Myc (top) or total cell lysates (bottom) were then analyzed for the indicated proteins. (D) Immunoblot for polymerase protein level in HEK293T cells overexpressing Flag-Abl and Flag-DDA1. (E) Immunoblot for polymerase protein level in HEK293T cells overexpressing Flag-Abl and knocking down DDA1. (F) Immunoblot for polymerase protein level in HEK293T cells overexpressing Flag-Abl, Myc-DDB1 wild type, and triple mutant (TM).

  • Fig. 5 Inhibition of c-Abl kinase activates HBV replication by stabilizing the level of HBV polymerase.

    (A) Strategy (see text for description). (B) HepG2 cells or (C) Huh7 cells were cotransfected with indicated plasmids and were treated with or without imatinib for 24 hours before harvest, whole-cell lysates were prepared for Western blotting (bottom), and capsid-associated viral DNAs were quantitated by real-time PCR (top). Mean copy number from cells only transfected with compHBV was set to 100% and compared with others (n = 3 per group). (D) HepG2 cells or (E) Huh7 cells were cotransfected with indicated plasmids and were treated with DMSO, MG132, or MLN4924 for 8 hours before harvest; whole-cell lysates were prepared for Western blotting (bottom); and capsid-associated viral DNAs were quantitated by real-time PCR (top). Mean copy number from cells treated with DMSO was set to 100% and compared with others (n = 3 to 4 per group). (F) HepG2 cells or (G) Huh7 cells were transfected with indicated siRNAs and plasmids, whole-cell lysates were prepared for Western blotting (bottom), and capsid-associated viral DNAs were quantitated by real-time PCR (top). Mean copy number from cells transfected with control siRNA was set to 100% and compared with others (n = 3 to 4 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 c-Abl inhibits HBV replication in vitro and in vivo.

    (A) Huh7 cells and (B) HepG2 cells were cotransfected with indicated plasmids, whole-cell lysates were prepared for Western blotting (bottom), and capsid-associated viral DNAs were quantitated by real-time PCR (top). Mean copy number from cells only transfected with compHBV was set to 100% and compared with others (n = 3 per group). (C) ICR mice were hydrodynamically injected with plasmid DNA, and capsid-associated HBV DNAs were purified from liver tissue. Mean copy number from liver of ICR mice hydrodynamic injected into sgCtrl was set to 100% and compared with others. Statistical significance compared with sgCtrl is noted by asterisks (lanes 1, 4, and 5: n = 4 per group; lane 2: n = 3; lane 3: n = 6). (D) Schematic model. c-Abl promotes CRL4Cdt2-mediated ubiquitination of HBV polymerase and further suppresses HBV replication. Imatinib promotes HBV reactivation through c-Abl kinase abrogation to down-regulate CRL4 activity, and bortezomib inhibits proteasome activity to protest the ubiquitination of HBV polymerase. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaau7130/DC1

    Fig. S1. Capsid-associated viral DNA level in the treatment of TKIs.

    Fig. S2. c-Abl kinase reduces the level of HBV polymerase.

    Fig. S3. c-Abl–CRL4Cdt2 reduces HBV polymerase by promoting its ubiqutination but not transcription level.

    Fig. S4. Inhibition of CRL4 E3 ubiquitin ligase enhances HBV replication by stabilizing viral polymerase.

    Fig. S5. Western blot–scanned films.

    Fig. S6. Western blot–scanned films.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Capsid-associated viral DNA level in the treatment of TKIs.
    • Fig. S2. c-Abl kinase reduces the level of HBV polymerase.
    • Fig. S3. c-Abl–CRL4Cdt2 reduces HBV polymerase by promoting its ubiqutination but not transcription level.
    • Fig. S4. Inhibition of CRL4 E3 ubiquitin ligase enhances HBV replication by stabilizing viral polymerase.
    • Fig. S5. Western blot–scanned films.
    • Fig. S6. Western blot–scanned films.

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