Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

  1. Jens Bukha
  1. aCopenhagen Hepatitis C Program, Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
  2. bDepartment of Pathology, Stanford University School of Medicine, Stanford, California, USA
  1. Fig 1
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    Fig 1

    Viability of E2-exchanged HCV recombinants. In vitro-generated RNA transcripts of H77C/JFH1Q1247L (TN-E2), H77C/JFH1Q1247L (J4-E2), and H77C/JFH1Q1247L (J6-E2) recombinants were transfected into Huh7.5 cells. The connecting lines represent the percent HCV NS5A-positive cells as determined by immunostaining at the indicated time points posttransfection (left y axis). The column bars show the infectivity titers for the E2-exchanged recombinants, which spread in culture following transfection (right y axis). Titer values are means of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 102.3 FFU/ml. *, measured virus supernatant under the limit of quantification.

  2. Fig 2
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    Fig 2

    HCV infectivity titers after transfection of the 1b-E2 H77C/JFH1V787A Q1247L recombinants with and without E2 stem region mutations. In vitro-generated RNA transcripts of H77C/JFH1V787A Q1247L (J4-E2) with or without V710F (A), H77C/JFH1V787A Q1247L (DH1-E2) with or without G706R (B), H77C/JFH1V787A Q1247L (DH5-E2) with or without S707L (C), and H77C/JFH1V787A Q1247L (J4-E2) with G706R or S707L, H77C/JFH1V787A Q1247L (DH1-E2) with S707L or V710F, and H77C/JFH1V787A Q1247L (DH5-E2) with G706R or V710F (D) were transfected into Huh7.5 cells. Virus production in the supernatant was measured on days 3, 6, 8, and 10 posttransfection. The previously developed H77C/JFH1V787A Q1247L recombinant was included as a positive control. Amino acid residues were numbered according to the polyprotein of the H77 reference sequence (GenBank accession number AF009606). All titer values are means of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 101.3 FFU/ml. *, samples analyzed in a separate experiment.

  3. Fig 3
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    Fig 3

    HCV infectivity titers after transfection of the 1b-E2 H77C/JFH1V787A Q1247L recombinants containing mutations identified outside the E2 stem region. In vitro-generated RNA transcripts of J4-E2I262F, J4-E2I262F T475A, DH1-E2M324T, DH5-E2V414A, and DH5-E2V414A L725F (see Table S1 in the supplemental material) were transfected into Huh7.5 cells. Virus production in the supernatant was measured on days 3, 6, 8, and 10 posttransfection. The previously developed H77C/JFH1V787A Q1247L recombinant was included as positive control. Amino acid residues were numbered according to the polyprotein of the H77 reference sequence (GenBank accession number AF009606). All titer values are means of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 101.3 FFU/ml.

  4. Fig 4
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    Fig 4

    Determination of intracellular core concentration at 4 h and 48 h posttransfection of transfected S29 cells, as a measure for HCV replication. Intracellular core protein synthesis was quantified using HCV core antigen ELISA on lysed S29 cells transfected with RNA transcripts of 1b-E2 H77C/JFH1V787A Q1247L recombinants with or without compensatory E2 mutations. For each recombinant, the core level measured at 48 h posttransfection is shown as a percentage of the 4-h core level (100%) in one replicate. Controls were H77C/JFH1V787A Q1247L (positive control), H77C/JFH1 (assembly defective), JFH1ΔE1/E2 (lacking expression of envelope proteins), and J6/JFH1-GND (replication defective).

  5. Fig 5
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    Fig 5

    Intracellular (A) and extracellular (B) infectivity titers of transfected S29 cells at 48 h posttransfection. In vitro transcripts of 1b-E2 H77C/JFH1V787A Q1247L recombinants with and without E2 compensatory mutations were transfected into S29 hepatoma cells lacking CD81. H77C/JFH1V787A Q1247L and JFH1ΔE1/E2 were included as positive and negative controls, respectively. All measurements are presented as mean values of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 100.7 FFU/well (A) or 101.3 FFU/ml (B). Samples with titers under the detection limit are indicated by an asterisk.

  6. Fig 6
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    Fig 6

    Influence of E2 exchange on particle assembly/release. (A) Core protein secretion of 1b-E2 H77C/JFH1V787A Q1247L recombinants with or without E2 compensatory mutations was quantified in S29 cells in three independent experiments at 48 h posttransfection using ELISA. Values are presented as mean percentages of the value for the positive control, H77C/JFH1V787A Q1247L (100%). Negative controls were H77C/JFH1 and JFH1ΔE1/E2. Statistical significance of core concentration differences was determined using the t test (a P value of <0.05 was considered significant), as stated in the text. (B) HCV release was measured by detection of extracellular core, E1, and E2 proteins in the virus supernatants collected at 48 h posttransfection of S29 cells, using SDS-PAGE and Western blotting with HCV-specific anti-core, anti-E1, or anti-E2 antibodies. Virus particles were concentrated on a 20% sucrose cushion from supernatant of cells transfected with 1b-E2 H77C/JFH1V787A Q1247L recombinants with and without compensatory E2 mutations. The negative control was JFH1ΔE1/E2.

  7. Fig 7
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    Fig 7

    Influence of compensatory E2 stem region mutations on HCV E1/E2 heterodimerization. Immunoblots of elution fractions of coimmunoprecipitation using CD81-LEL-GST- or GST-tagged coated beads on lysates from transfected 293T cells (A) and of input fractions of 293T cell lysates prior to coimmunoprecipitation using anti-E1 and anti-E2 specific antibodies (B) are shown. An H77C E1/E2 positive control was transfected in duplicate and served as a specificity control, as co-IP was conducted using both CD81-LEL-GST-coated beads (H77 E1/E2) and GST-tagged coated beads only (H77 E1/E2 GST). The ΔE1/E2 construct (phCMV-ires, in which the partial E1/E2 HCV sequence was deleted) was included as an E1/E2 negative control. β-Actin served as loading control.

  8. Fig 8
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    Fig 8

    Density analysis of HCVcc particle infectivity (A), total core amount produced (B), and specific infectivity (C) of 1b-E2-exchanged H77C/JFH1V787A Q1247L recombinants with or without compensatory E2 mutations: H77C/JFH1V787A Q1247L (J4-E2) with or without V710F, H77C/JFH1V787A Q1247L (DH1-E2) with or without G706R, and H77C/JFH1V787A Q1247L (DH5-E2) with or without S707L. Supernatants harvested from S29 cells at 48 h posttransfection were ultracentrifuged in a 10 to 40% iodixanol gradient, and fractions were analyzed. The transfection efficiency was monitored in all cultures using NS5A-specific immunostaining prior to ultracentrifugation. The positive control was H77C/JFH1V787A Q1247L. In panel A, quantified titers are presented as the mean values of triplicates, and the lower limit of detection is 102.0 FFU/ml. In panel C, the peak titer fraction for E2-exchanged recombinants with compensatory mutations is indicated with red arrows.

  9. Fig 9
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    Fig 9

    HCV particle stability at 37°C, analyzed during a period of 32 h. In vitro-generated RNA transcripts of DH5-E2 and DH5-E2S707L were transfected into S29 cells for production of HCVcc. At 48 h posttransfection, particle-containing supernatant was harvested, aliquoted, and incubated at 37°C. At the indicated time points, supernatant was transferred to −80°C, followed by subsequent infectivity titration in naïve Huh7.5 cells. Titer results are presented as percent infection compared to the 0-h infectivity level. Values shown are means from three independent assays, with error bars displaying standard errors of the means (SEM) between assays; for each independent assay, titrations for all time points were in triplicates.

  10. Fig 10
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    Fig 10

    HCVcc particle binding assay on 1b-E2-exchanged recombinants with or without stem region mutations. In vitro transcripts of 1b-E2 H77C/JFH1V787A Q1247L recombinants with and without E2 compensatory mutations were transfected into S29 cells for production of HCVcc. At 48 h posttransfection, particle-containing supernatant was harvested and an inoculum was applied to naïve Huh7.5 cells for 2 h at 4°C to allow binding but not entry of the HCVcc particles. Following incubation, cells were washed and lysed, and cell-bound core protein was quantified using core ELISA. Results are presented as cell-bound core protein in percentage of the core input for each isolate. All measurements are presented as mean values from duplicate experiments, with error bars displaying standard errors of the means (SEM). Controls were H77C/JFH1V787A Q1247L (positive control), and JFH1ΔE1/E2 (lacking envelope proteins).

  11. Fig 11
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    Fig 11

    Dose-dependent inhibition of DH5-E2 with or without S707L binding to the CD81 receptor using CD81 specific antibody. In vitro-generated RNA transcripts of DH5-E2 and DH5-E2S707L were transfected into S29 cells for production of HCVcc. At 48 h posttransfection, particle-containing supernatant was harvested and DH5-E2 with or without S707L inoculum was applied to naïve Huh7.5 cells which had been incubated with anti-CD81 antibody at concentrations ranging from 0.004 to 2.5 μg/ml for 1 h preinfection. Results are presented as the means of 4 replicates at each antibody concentration, with error bars representing SEM. Percent inhibition was calculated relative to infectivity titers of 6 replicates of virus only. Open symbols represent percent inhibition relative to virus only using an isotype-matched negative control at a concentration of 2.5 μl/ml.

  12. Fig 12
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    Fig 12

    Effect of compensatory E2 mutations on chimeric HCV pseudoparticle entry. (A) Infectivity of HCVpp was detected by luciferase reporter gene expression. Infectivity is presented in relative light units (RLU) obtained in 20 μl of cell lysate from a representative experiment. Error bars show the standard errors of the means (SEM) of 8 replicates. (B and C) Immunoblots of concentrated HCVpp (B) and HCVpp 293T cell lysate (C) using anti-E1 and anti-E2 specific antibodies, originating from the experiment presented in panel A. The H77C E1/E2 positive control was transfected in duplicates and served as an intraexperiment control. The ΔE1/E2 construct (phCMV-ires in which the HCV sequence was deleted) was included as a negative control. Anti-β-actin and anti-Gag MLV (CA) were included as loading controls.

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This Article

  1. Accepted manuscript posted online 14 November 2012, doi: 10.1128/JVI.00684-12 J. Virol. vol. 87 no. 3 1385-1399
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