Volume 124, Issue 7, Pages 1809-1820 (June 2003) Relevance of hepatitis B core gene deletions in patients after kidney transplantation  C.-Thomas Bock, Boris Buerke, Hans L Tillmann, Frank Tacke, Volker Kliem, Michael P Manns, Christian Trautwein  Gastroenterology  Volume 124, Issue 7, Pages 1809-1820 (June 2003) DOI: 10.1016/S0016-5085(03)00396-2

Figure 1 Wt-HBV and mutant HBV-core gene deletions after kidney transplantation. (A ) PCR amplification of the coding region of the HBV core gene. The HBV core region of patients 1–30 after kidney transplantation were analyzed from serum. The PCR products were analyzed on a 1% agarose gel. Size variations of the PCR product revealed core gene deletions. The gel depicts 6 of 10 core gene deletions (lanes 2–7) found in the serum of HBV-infected patients after kidney transplantation. Lane 1 shows the molecular weight marker. (B) Schematic description of wt (upper lane, HBV subtype adw2) and patients’ core gene deletions (patients 1–10). The deletions are shown as a dark heavy line. Positions of the deletions are depicted as amino acids (B; AA; core-methionine = AA 1). Functional regions in the preC/core gene are denoted (B; upper lane). NLS, nuclear localization signal. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 2 Transcriptional and replication activity of core gene deletion mutants. (A ) Northern blot analysis was performed with total RNA derived from HuH7 cells transfected with HBV wt and preC/C-mutant constructs. Twenty μg RNA from mock-transfected cells (pBluescript; lane 2) and cells transfected with wild-type HBV (pHBV2; lane 1) or mutant HBV (patients 1–10; lanes 3–12) plasmids were separated on a 1% gel and transferred to Nylon membranes. HBV-specific RNA was detected with a [32P]-labeled HBV probe. Position of the HBV-specific RNAs is shown at the right. Size markers were 18S and 28S RNA at the left. (B) HuH7 and HepG2 cells were transfected with HBV replication-competent constructs. Vector control (pBS, lane 2), wt (pHBV 2.0; lane 1) and core gene deletion mutants (patients 1–10, lanes 3–12) were used. The amount of newly synthesized progeny DNA was determined from cytoplasmic HBV capsid-like particles using anti-HBc antibodies for immunoprecipitation from 106 cells. HBV DNA was visualized by dot blot analysis using a [32P]-labeled HBV probe. (C ) Analysis of encapsidated pregenomic HBV-RNA (3.5 kb) from transiently transfected HuH7 and HepG2 cells with wt HBV (pHBV2; lane 2), negative control vector (lane 1), and core gene deletion mutants (patients 1–10; lanes 3–12) was performed using immunoprecipitated cytoplasmic HBV capsid-like particles. Isolated pregenomic HBV RNA was separated on 1% agarose gel and visualized by Northern blot analysis using a [32P]-labeled HBV probe. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 3 Decreased levels of HBsAg and HBeAg in the supernatant of HuH7 and HepG2 cells transfected with HBV core gene deletion constructs. (A, B) Transfection experiments in HuH7 and HepG2 cells were performed using wt and mutant HBV core-gene deletion constructs. The amount of (A ) HBsAg and (B) HBeAg in the cell culture supernatants was determined by micro enzyme immuno assay 6 days after transfection. The different HBV core gene deletion constructs (A and B, lanes 1–10; patients 1–10) are depicted. HBeAg and HBsAg expression as determined by the wt HBV construct (A and B, pHBV2) was set to 100%. The concentrations derived in the parallel transfection experiments as indicated were calculated accordingly. Error bars indicate SDs. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 4 Intracellular localization of wt and mutant HBV core protein. (A, B) Immunofluorescence experiments using HBcAg staining were performed in (A ) cells transfected with HBV-replication competent and (B) pcDNA-expression vectors using mock control (A and B; empty vector; panel 1), wt HBV (A and B; pHBVwt; panel 2), or mutant (A and B; patients 1–10; panels 3–11) HBV core gene deletion plasmids. HBcAg was visualized using anti-HBcAg polyclonal antibodies and Cy3-conjugated secondary antibodies. Nuclei of the same set of cells were stained using the DNA staining dye DAPI. Magnification 70×. (C ) Intracellular localization of GFP-HBV-core fusion proteins visualized by fluorescence microscopy. HuH7 cells were transfected with pcDNA-EGFP control (C; mock control), pcDNA-GFP-core gene wt (C; pHBVwt), and mutant constructs (C, patients 1–10, panels 1–10). Mutants 5 and 7 showed an irregular nuclear localization compared with wt HBV constructs. Magnification 200×. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 5 Complementation of wt HBV with mutant 5 and 7 constructs. (A, B) Different amounts (1 + 5 μg) of the wt HBV 2.0 construct were either infected alone or a constant amount (1 μg) of the wt HBV construct was cotransfected with increasing amounts (1 + 5 μg) of the core mutant HBV 2.0 constructs (patients 5 + 7). (A ) In the supernatant HBsAg expression was determined or (B) the amount of progeny DNA was quantitated by dot blot analysis. (C ) The wt HBV 2.0 construct (3 μg) was cotransfected with increasing amounts (1 + 3 μg) of the core mutant HBV 2.0 constructs (patients 5 + 7). HBV particles were PEG precipitated from the supernatant and further processed to receive viral progeny DNA. PCR was performed with core primers to differentiate between the wt and mutant core genome. The position of the wt and mutant core PCR product is indicated. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 6 Complementation of mutant 5 and 7 constructs with wt HBV. (A ) Progeny HBV-DNA transcomplementation experiments were performed by transfection experiments. Three μg of core-deletion constructs of patients 5 and 7 (panels 1–2) were cotransfected with 0 ng (lane 1), 1 μg (lane 2), and 5 μg (lane 3) wt HBV pHBV2 plasmids, respectively. HBV capsids were immunoprecipitated from cells after 6 days using anti-HBcAg antibodies. Progeny HBV DNA was detected by dot blot analysis. (B) Intracellular localization of wt HBV-transcomplementated patients 5 and 7 mutant core protein. HuH7 cells were transfected with 3 μg of the wt HBV 2.0 construct (HBV-WT) or 3 μg each of the wt HBV 2.0 and the respective mutant HBV 2.0 construct derived from patient 5 and 7. Immunofluorescence experiments with anti-HBcAg antibodies and Cy3-conjugated secondary antibodies were performed. DAPI staining of the same set of cells showed corresponding cell nuclei. Magnification ×70. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)

Figure 7 Core gene deletions 5 and 7 were associated with a more severe course of chronic HBV infection. (A) Time course of serum transaminase values of patients 1–10 (triangles) with the exception of patients 5 (circles) and 7 (rhombuses) after kidney transplantation. Patient 5 died 5 years after kidney transplantation. (B) Liver function as determined by CHE values of patients 1–10 (triangles) with the exception of patients 5 (circles) and 7 (rhombuses) after kidney transplantation. Gastroenterology 2003 124, 1809-1820DOI: (10.1016/S0016-5085(03)00396-2)