Volume 131, Issue 3, Pages 765-780 (September 2006) Functional Analysis of Complex Hepatitis B Virus Variants Associated With Development of Liver Cirrhosis  Stefanie Märschenz, Anne–Sophie Endres, Anja Brinckmann, Tilman Heise, Glen Kristiansen, Peter Nürnberg, Detlev H. Krüger, Stephan Günther, Helga Meisel  Gastroenterology  Volume 131, Issue 3, Pages 765-780 (September 2006) DOI: 10.1053/j.gastro.2006.07.008 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Analysis of HBV wild-type (wt) genomes wtD and wtA and 15 clones isolated from the patient’s serum (S) and liver (L) after transfection into HuH7 cells. For detailed mutation pattern see Table 1. Variants with C gene deletion were cotransfected with core expression plasmid pCore for complementation of core protein and restoration of replication competence. NC, negative control, only pZero transfected. (A) Southern blots of replicative intermediates from intracellular core particles. HBV marker: 1:1000 dilution of products of HBV full-length genome asymmetric PCR (containing 3.2-kb double-strand [ds] and single strand [ss] DNA fragments). The collection date of the sample the clones were derived from is indicated below the blots. Bottom: Quantitative evaluation of the band intensities of the shown blots relative to wtD transfected alone by TINA software. For the 5 clones analyzed in detail (bold and underlined, dark shaded bars), mean values and standard deviations of at least 6 independent experiments are shown. For all other clones, only 1 experiment was performed. (B) Northern blots of total RNA. pg, pregenomic RNA; preC, precore mRNA. Actin RNA was used for loading control. Bottom: Quantitative evaluation of the level of the pg/preC RNA relative to wtD. For the 5 clones analyzed in detail (bold, dark shaded bars), mean values and standard deviation of at least 3 independent experiments are shown. For all other clones, only 1 experiment was performed. (C) HBsAg and HBeAg levels in the cell culture supernatant (diluted 1:10) relative to wtD levels detected by ELISA. Mean values of at least 4 transfections. pCore, only pCore was transfected. Note that transfection with pCore causes low levels of HBeAg detection (see lane pCore), also in samples of C gene deletion variants cotransfected with pCore. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 Viral population in correlation to the clinical course of the patient and variants analyzed in detail. (A) Clinical course during follow-up: alanine aminotransferase (ALT) levels and viral load, therapy with famciclovir (FCV) and lamivudine (3TC), and time points of first diagnosis of LC and combined liver/renal transplantation (LTx/RTx) are given. Isolation time points of the variants analyzed in detail are indicated on top. (B) Presence and frequency of variant types A, E, and J in the viral population during follow-up until combined RTx and LTx are shown schematically. Variant types A–J occurring in this patient were previously defined.3 The definitions of types A, E, and J are given on the right. Variant types E and J accumulated and represented the main viral population during LC. Del, deletion; ins, insertion; mut, mutation. (C) Schematic presentation of the variant genomes analyzed in detail. The HBV genome organization is schematically shown on top. Main viral mutations (deletions, open boxes; insertions, small triangles) that were used for variant type definition and nucleotide changes (open ellipse) in Cp are indicated on the genomic sequence (horizontal line) of each variant. Major changes in expected gene expression are shown below the genome. Predictably expressed protein sequences are shown by an open box, whereas predictably nonexpressed sequences because of premature stop codons (star) or a start codon mutation (x) are shown by dotted lines. Variant S15 has a frameshift (fsh) in the C gene starting at amino acid (aa) 36. Collection dates of the samples the clones were isolated from are indicated on the left, the variant type according to Preikschat et al3 is indicated on the right. All clones carry further point mutations throughout the whole genome (Table 1). The sequences have been sent to GenBank and assigned the following accession numbers: DQ788725-DQ788729. SHBs, MHBs, LHBs: small, middle, and large HBV surface protein, respectively. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 Analysis of HBV RNA transcripts. HBV RNA was analyzed 4 days after transfection of HuH7 cells with wt genomes or variant HBV DNA. pg, pregenomic RNA; preC, precore mRNA. (A) Exemplary Northern blot of total RNA detected with a full-length HBV RNA probe. Marker, Dig-marker II (Roche). Actin RNA was detected as loading control. (B) Quantitative evaluation of the 3.5-kb pg/preC-RNA signals in Northern blots relative to wtD by TINA software. Mean values and standard deviations of 3 independent experiments. (C) Primer extension analysis of preC-mRNA and pgRNA performed with total RNA. A sequencing reaction of a wt (genotype A) genome using the same primer served as marker (not shown). Top: Gel picture of 1 representative experiment is shown. NC, negative control, only vector pZErO transfected. Bottom: Quantitative evaluation of the signals with TINA software. Mean and standard deviations of preC-mRNA to pgRNA ratios of 2 to 14 independent experiments are shown. (D) Quantitative evaluation of preS1-mRNA and preS2/S-mRNA signals in Northern blots of 3 independent experiments by TINA software. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 4 Analysis of major spliced pregenomic RNA type 1 (SP1) and corresponding replicative intermediates. (A) Schematic presentation of the unspliced pregenomic (pg) RNA, the major spliced RNA SP1 with the splice donor site (SD) at nt 2447 (genotype A: nt 2453), and the splice acceptor site (SA) at nt 489 and the 2 subgenomic surface mRNA. The positions of the Northern blot probe for SP1 detection spanning nt 2021–2755 and thus not hybridizing with subgenomic RNA as well as of the PCR primers (arrows) are indicated. (B) Northern blot of total RNA of HBV-transfected HuH7 cells. HBV pg/precore (preC) and SP1 signals are indicated. Detection of SP1 was not hampered by the C gene deletion as verified with hybrid genome S1/L1-C (data not shown) (C) Ratio of SP1 to pgRNA/preC mRNA signals in Northern blot reproduced in 3 independent experiments. (D) Replicative intermediates from cytoplasmic core particles were isolated after transfection of wtD, wtA, wt-like S1, and complex variants + pCore (analogous to replication analysis) and subjected to PCR detection with primers binding upstream and downstream of the SP1 splice sites. Bands of unspliced and SP1-derived DNA in a 1% agarose gel are shown. NC, negative control. (E) Ratio of SP1-derived to unspliced DNA signals (see D) reproduced in 2 independent experiments. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 5 Analysis of replication. HuH7 cells were transfected with viral DNA and harvested after 4 days. WtD, wtA, S1, or variant (var) DNA was transfected alone (2 μg) or with pCore (1.6 μg/2 μg of HBV DNA). Alternatively, variants or variant pools were cotransfected with wtD in a 2:8 ratio (total 2 μg). Variant pools contain equal DNA amounts of variants representative of the entire HBV population found in serum of 05/96 (pool 1: 3 clones including S24, Figure 1, Table 1) or in the liver of 07/96 (pool 2: 6 clones including L1, Figure 1, Table 1). (A) Representative Southern blots of HBV replicative intermediates from intracellular core particles. ds, double strand; NC, negative control, only vector pZErO transfected; ss, single strand. (B) Replication level of wt genomes, S1, and variants with or without pCore relative to wtD alone determined by quantitative evaluation of Southern blot signals by TINA software. Means and standard deviations of at least 6 independent experiments. (C) Quantitative evaluation of total variant (or pool) + wtD replication after cotransfection in a 2:8 ratio determined by Southern blot. Signal intensities relative to the wtD signal. Mean values and standard deviations of at least 3 (for S24 + wtD, only 2) independent experiments are shown. (D) Enrichment of variant (or pool) relative to wtD (x-fold) compared with the originally transfected proportion. The percentages of variant (or variant pool) and wtD nucleic acids in cytoplasmic core particles or supernatant were determined by pyrosequencing. Enrichment = (% variant/% wtD) nucleic acid after transfection/(% variant/% wtD) transfected DNA. It was assured by pyrosequencing of nuclear DNA that the percentages of DNA in the nucleus correspond to the percentages of transfected wtD and variant DNA (80% and 20%, respectively, data not shown). Mean and standard deviations of at least 3 independent experiments. Top: intracellular, core-particle-associated pregenomic RNA (reverse transcribed prior to pyrosequencing). Middle: intracellular replicative intermediates. Bottom: particle-associated progeny DNA in cell culture supernatant. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 6 Analysis of secreted HBeAg and intracellular core protein 4 days after transfection of HuH7 cells with wtD, wtA, wt-like S1, or variant DNA. (A) HBeAg levels in cell culture supernatant (diluted 1:10) relative to wtD levels detected by ELISA. Mean values and standard deviations of at least 4 experiments. NC, negative control, only vector pZErO transfected. (B) Immunocytochemical staining of HBV core protein with polyclonal anti-coreSN antibody. Cells were counterstained with hematoxylin (original magnification, ×100). Exemplary positive cells are marked by an arrow. (C) Double-immunofluorescence staining of core protein with polyclonal anti-coreSN antibody and of cytoplasm with anti-Hsp70 antibody. Confocal laser scanning microscopy was performed of stained cells transfected with wtD, with variant + pCore, and with variant + wtD in a 2:8 ratio. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 7 Analysis of secreted HBsAg and intracellular HBV surface proteins 4 days after transfection of HuH7 cells with wtD, wtA, wt-like S1, or variant DNA. NC, negative control; only vector pZErO transfected. (A) HBsAg levels in cell culture supernatant (diluted 1:10) relative to wtD levels detected by ELISA. Mean values and standard deviations of at least 4 experiments. (B) Western blots of cell lysate detecting HBV surface proteins with primary antibodies MA18/7 (anti-preS1) and polyclonal anti-HBs. Detection was carried out with peroxidase-coupled secondary antibodies and chemiluminescence substrate in a CCD camera. Detection with anti-β-actin antibody was performed as loading control. Marker, 60 ng purified HBsAg. p24, p39, gp27, gp33, gp42, and ggp36 denote unglycosylated and singly or doubly glycosylated surface proteins. (C) Immunocytochemical staining of HBV surface proteins with primary monoclonal antibodies MA18/7 (anti-preS1), F6 (anti-preS2), and RF18 (anti-S). Cells were counterstained with hematoxylin (original magnification, ×100). Exemplary positive cells are marked by an arrow. (D) Double-immunofluorescence staining of surface proteins with primary antibodies MA18/7 (top set), F6 (bottom left set), RF18 (bottom right set), and of endoplasmic reticulum (ER) with anti-calnexin antibody. Confocal laser scanning microscopy was performed. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 8 Analysis of hybrid genomes. Nucleic acids were analyzed 4 days after transfection of HuH7 cells with HBV DNA. Mean values and standard deviations are shown in the graphs. (A) Schematic presentation of the hybrid variants. For generation of hybrid genomes S1/L1-Cp, S1/L1-C, and S1/L1-C/Cp, the Cp and/or C gene of wt-like genome S1 were replaced by the corresponding regions of L1. (B) Analysis of transcription. A representative Northern blot of total RNA performed as described in Figure 3 is shown. The results were confirmed by 2 additional independent experiments. (C) Ratio of preC-mRNA to pgRNA as determined by quantitative signal evaluation of 2–7 independent primer extension analyses. (D) Level of HBeAg in cell culture supernatant (1:10 dilution) of 3 independent experiments detected by ELISA. (E and F) Analyses of replicative intermediates from cytoplasmic core particles of at least 3 independent experiments performed as described in Figure 5. (E) Left: Representative Southern blot of replicative intermediates after transfection of S1 and S1/L1-Cp alone, as well as L1, S1/L1-C, or S1/L1-C/Cp with pCore. Right: Quantitative evaluation of Southern blot signals relative to S1. (F) Enrichment of complex variant L1 or hybrid variants relative to wtD (x-fold) as determined by pyrosequencing after cotransfection of L1 or hybrid variants L1-C or L1-C/Cp with wtD in a 2:8 ratio. Gastroenterology 2006 131, 765-780DOI: (10.1053/j.gastro.2006.07.008) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions