Volume 136, Issue 1, Pages 168-176.e2 (January 2009) In Vitro Characterization of Viral Fitness of Therapy-Resistant Hepatitis B Variants  Stéphanie Villet, Gaëtan Billioud, Christian Pichoud, Julie Lucifora, Olivier Hantz, Camille Sureau, Paul Dény, Fabien Zoulim  Gastroenterology  Volume 136, Issue 1, Pages 168-176.e2 (January 2009) DOI: 10.1053/j.gastro.2008.09.068 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Evolution of HBV variants selection during anti-HBV therapy. In the first part of the figure are represented previous results of polymerase gene clonal analysis performed on HBV DNA isolated from patient sera.8 Each diagram corresponds to HBV quasispecies isolated from one patient's serum during lamivudine monotherapy or lamivudine plus adefovir plus HB Ig tritherapy. At the different time points, the co-existence of different mutants was observed, each represented by a specific color or motif. The study was focused on the last 2 time points, illustrating the selection of mutant 1 among HBV quasispecies. In the second part of the figure are listed mutations in RT and S-associated domains for the 4 main mutants. Specific RT or S domain mutations are highlighted in bold. The other mutations are the consequences of mutations in the overlapping gene. Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 In vitro replicative capacity of the selected resistant HBV mutant 1 is the highest in Huh-7 cells in the presence of both lamivudine plus adefovir. Huh-7 cells were transfected transiently with each HBV mutant or wt HBV, and treated with high concentrations of lamivudine (100 μmol/L) and adefovir (100 μmol/L) for 5 days. HBV DNA from intracellular core particles then were purified following the protocol previously described.18 Intracellular HBV DNA was subjected to Southern blot analysis with an anti-HBV probe, and then quantified by Phosphorimager analysis. Mutant replication capacity is reported to wt replication in arbitrary units (AUs). Standard deviations were obtained from 3 independent experiments performed in duplicate. Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Synthesis and accumulation of intracellular envelope proteins are similar between mutant 1 and wt HBV. Huh-7 cells were transfected transiently with each HBV mutant, wt HBV, or not transfected. (A) Western blot analysis. Cells were lysed 9 days after transfection, and proteins were separated by electrophoresis in 12% acrylamide gel. After transfer to a nitrocellulose membrane, S, M, and L proteins were visualized after immunodetection with a mixture of rabbit anti-S (1:500 dilution) and mouse anti–Pre-S2 antibody (1:1000 dilution). T(−) corresponds to nontransfected Huh-7 cells. (B) Immunofluorescence microscopy. Cells were fixed 5 days after transfection and stained first with a mouse anti–Pre-S2 or a rabbit anti-S antibody, and second with Alexa Fluor 555–conjugated goat anti-mouse or anti-rabbit antibody. Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Secretion of HBV particles with mutant 1 is close to wt HBV and higher than the other mutants. Huh-7 cells were transfected transiently with each HBV mutant, wt HBV DNA, or not transfected. Supernatant was harvested at days 5, 7, 9, and 12 posttransfection and concentrated by ultracentrifugation. (A and B) Western blot analysis. Equivalent volumes of supernatant for each mutant were loaded on a 12% acrylamide gel. After transfer to a nitrocellulose membrane, S, M, and L proteins were visualized by immunodetection with a (A) mouse anti–Pre-S2 (1:1000 dilution) or a (B) rabbit anti-S (1:500 dilution) antibody. T(−) corresponds to nontransfected Huh-7 cells and T(+) to purified Dane particles. (C) Immunoprecipitation of Dane particles. Equivalent volumes of supernatant for each mutant were immunoprecipitated with a mouse anti–Pre-S1 antibody and then submitted to Dot blot analysis with an anti-HBV probe. Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Dane and subviral particles produced by mutants 1, 2, and 4 have a correct morphology. Viral suspensions were adsorbed on nickel grids and either directly stained with phosphotungstic acid or stained for immunogold analyses. When micrographs were obtained after immunogold experiments, the primary antibody used is mentioned at the upper left corner (anti-S, anti–Pre-S2, or anti–Pre-S1 antibody). Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 A higher level of secretion and infectivity of HDV particles is evidenced for selected mutant 1. Huh-7 cells were cotransfected transiently with both pTriex-HBV mutant or wt and pSVLD3 plasmid expressing HDV genome. Supernatants were collected at days 5, 7, and 9, and concentrated by ultracentrifugation. (A) Replication and secretion of HDV genome. Five micrograms of cellular RNA (Cell) and equivalent volumes of supernatant (Sup) for each mutant were loaded on a 1.2% agarose gel. After loading control and transfer to a nitrocellulose membrane, HDV genome was visualized after Northern blot with an anti-HDV probe. T(−) corresponds to pSVLD3-transfected cells. (B) Secretion of HDV particles. Equivalent volumes of supernatants were immunoprecipitated for each mutant with a mouse anti–Pre-S1 antibody and then submitted to Dot blot analysis with an anti-HDV probe. (C) Infectivity of HDV particles. Three × 105 differentiated HepaRG cells were exposed to HDV particles coated by HBV wt or mutant envelope proteins for 20 hours (multiplicity of infection, 250). Eight days postinfection, cellular RNA was extracted and loaded on a 1.2% agarose gel. After transfer to a nitrocellulose membrane, HDV RNA was detected by Northern blot analysis with an HDV-RNA–specific probe. Mutant infectivity is expressed as a percentage of wt infectivity. Standard deviations were obtained from 6 independent experiments. Gastroenterology 2009 136, 168-176.e2DOI: (10.1053/j.gastro.2008.09.068) Copyright © 2009 AGA Institute Terms and Conditions