Incorporation of primary patient-derived glycoproteins into authentic infectious hepatitis C virus particles

Authors

  • Juliane Doerrbecker,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Martina Friesland,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Nina Riebesehl,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Corinne Ginkel,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Patrick Behrendt,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
    2. Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Germany
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  • Richard J.P. Brown,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
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  • Sandra Ciesek,

    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
    2. Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Germany
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  • Heiner Wedemeyer,

    1. Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Germany
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  • Christoph Sarrazin,

    1. Klinikum der Johann Wolfgang Goethe-Universität, Germany
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  • Lars Kaderali,

    1. Institute for Medical Informatics and Biometry, Medical Faculty, Technische Universität Dresden, Germany
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  • Thomas Pietschmann,

    Corresponding author
    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
    • Address reprint requests to: PD Dr. Eike Steinmann or Prof. Dr. Thomas Pietschmann, Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany. E-mail: Eike.Steinmann@twincore.de or Thomas.pietschmann@twincore.de; fax: +49 2200 27139.

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    • These authors contributed equally.

  • Eike Steinmann

    Corresponding author
    1. Institute for Experimental Virology, Twincore, and Hannover Medical School Hannover, Germany, and Helmholtz Centre for Infection Research, Braunschweig, Germany
    • Address reprint requests to: PD Dr. Eike Steinmann or Prof. Dr. Thomas Pietschmann, Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany. E-mail: Eike.Steinmann@twincore.de or Thomas.pietschmann@twincore.de; fax: +49 2200 27139.

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    • These authors contributed equally.


  • Potential conflict of interest: Dr. Pietschmann consults for Biotest and Janssen.

  • E.S. was supported by the DFG (STE 1954/1-1) and an intramural young investigator award of the Helmholtz Centre for Infection Research. T.P. was supported by grants from the DFG (PI 734/2-1) and the Helmholtz Association (SO-024) and the i-Med initiative.

Abstract

The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. Conclusion: This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion. (Hepatology 2014;60:508–520)

Abbreviations
E-I

encephalomyocarditisvirus internal ribosomal entry site

HCV

hepatitis C virus

HCVcc

hepatitis C virus cell culture

HCVpp

hepatitis C virus pseudoparticles

HCVTCP

hepatitis C virus trans-complemented

GT

genotype

JFH1

Japanese fulminant hepatitis-1

P-I

poliovirus internal ribosomal entry site

qRT-PCR

quantitative real time-polymerase chain reaction

TCID50

tissue culture infectious dose 50

Globally, an estimated 160 million people are chronically infected with hepatitis C virus (HCV)[1] and are therefore at a high risk for developing severe liver disease including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma.[2] Due to error-prone RNA replication, HCV is a genetically highly variable virus and based on phylogenetic analyses viral isolates are grouped into seven genotypes, which differ between each other by more than 30% at the nucleotide level.[3] This genetic variability facilitates immune evasion and contributes to viral persistence. Importantly, genotype (GT)-dependent viral determinants influence both treatment response and the natural course of hepatitis C, since GTs 1 and 4 are more difficult to treat with interferons compared to GTs 2 and 3 and as GT 3 is associated with liver steatosis.[4] Unfortunately, primary patient-derived HCV replicates poorly in cell culture, precluding in-depth assessment of viral functions causing these differences.

The development of in vitro cell-culture systems, including subgenomic replicons for GT 1, 2 and more recently for GT 3 and 4,[5-8] HCV pseudoparticles,[9, 10] and ultimately the Japanese fulminant hepatitis-1 (JFH1)-based infection system[11-13] have allowed detailed investigation of many aspects of the viral life-cycle and facilitated evaluation of antiviral compounds.[14] More recently, additional GT 2 and GT 1 infectious culture systems including JFH-2,[15] J6cc, J8cc,[16] H77-S,[17] and TN[18] have been reported. Finally, construction of JFH1-chimeras has permitted analysis of virus assembly and HCV entry across all HCV genotypes.[11, 19, 20] However, these models are limited to distinct, more or less representative clonal HCV sequences which may not adequately reflect the entire spectrum of functional traits of primary HCV in vivo.

Nevertheless, using these systems we and others have recently reported HCV isolate-dependent use of claudin and occludin family members for infection, thus highlighting strain-specific functional differences between HCV isolates during cell entry.[21, 22] These differences are caused by the viral glycoproteins E1 and E2 that mediate receptor interactions critical for cell entry and infection of hepatocytes. In parallel, these viral proteins are the main targets for neutralizing antibodies that exert strong immune pressure, thus rendering the glycoproteins most variable among all HCV proteins.[3] However, to what extent divergent glycoprotein function and HCV receptor use contributes to viral immune escape and differential course of infection between individuals is unknown. Therefore, to facilitate individualized studies of HCV glycoprotein function, in this work we aimed at developing cell culture systems for assessment of functional traits of patient-derived primary glycoproteins in the context of authentic liver-cell derived HCV particles.

Materials and Methods

Ethics Statement

The study was approved by the Ethics Committee of the Hannover Medical School. Patients provided written informed consent.

Cell Culture and Cell Lines

Huh-7.5, Huh-7.5.1, and 293T cells were cultured in Dulbecco's modified minimal essential medium (DMEM, Life Technologies) supplemented with 1× nonessential amino acids (Invitrogen), 100 μg/mL streptomycin (Invitrogen), 100 IU/mL penicillin (Invitrogen), and 10% fetal bovine serum (DMEM complete) at 37°C and 5% CO2. Packaging cell lines Huh-7.5[C][p7NS2]J6, Huh-7.5[C][p7NS2]H77, Huh-7.5[C][p7NS2]H77/V787A, Huh-7.5.1[C][p7NS2]J6, and Huh-7.5.1[C][p7NS2]H77/V787A ectopically expressing viral proteins were created by lentiviral gene transfer as described recently.[23] Gene transfer involved vectors encoding a blasticidin-S deaminase resistance gene, and therefore cells were selected by addition of blasticidin at a dose of 5 μg/mL.

Phylogenetic Analysis

E1E2 nucleotide alignments were generated using the ClustalX algorithm implemented in MEGA5.[24] Nucleotide sequences were aligned according to encoded amino acid sequences, to ensure maintenance of the overlying open reading frame. Alignments were manually adjusted and gap-stripped prior to phylogenetic reconstruction to avoid erroneous comparison of nonhomologous sites. The genotypes of recovered E1E2 sequences were confirmed by phylogenetic analyses, using the maximum likelihood method implemented in PhyML 3.0.[25] Robustness of major phylogenetic groupings was assessed by way of the bootstrap approach using 1,000 replicates, and presented as percentages.

Plasmids and Viral Genomes

The pWPI derivates pWPI-core-J6_BLR, pWPI-core-H77_BLR, pWPI-p7NS2-J6_BLR, pWPI-p7NS2-H77_BLR pWPI-p7NS2-H77/V787A_BLR were generated as previously described.[26] The plasmids pFK PI-EI-NS3-NS5B/JFH1 and pFK PI-G-Luc-EI-NS3-NS5B/JFH1 have been described elsewhere.[23, 26] The basic helper replicon vector was engineered with a poliovirus-derived internal ribosome entry site (IRES) (designated P-I) downstream of the JFH1-derived 5-nontranslated region (5′NTR) (nucleotides 1 to 341 of JFH1) and separated from the HCV 5′NTR by a spacer region of 72 nucleotides. The design of the Renilla luciferase reporter replicon is according to the R2a Jc1 construct described before.[27] In brief, the Renilla luciferase is fused to the NS3-NS5B coding region of JFH1 by way of the foot-and-mouth disease virus 2A peptide coding region that liberates the reporter from the downstream HCV proteins. The ΔCE1E2 genes of isolates H77 (GT 1a), Con1 (GT 1b), J4 (GT 1b), JFH1 (GT 2a), J6CF (GT 2a), J8 (GT 2b), S52 (GT 3a), ED43 (GT 4a), SA13 (GT 5a), Hk6a (GT 6a), QC69 (GT 7a), and plasma derived isolates were transferred into the first cistron of pFK PI-EI-NS3-NS5B/JFH1 by polymerase chain reaction (PCR)-based insertion. Further details regarding the cloning strategies and exact nucleotide sequences can be obtained upon request.

In Vitro Transcription, Transfection, and Production/Titration of Trans-Complemented HCV Particles

HCV trans-complemented particles were produced as described previously.[23] For infectious particle quantification, virus-containing culture fluids were harvested at indicated timepoints and filtered through a 0.45-μm pore size filter. Viral infectivity of cell-free supernatants was determined by infection of naïve Huh-7.5 target cells. Infectious particle amounts were quantified by a slightly modified limiting dilution assay on naïve Huh-7.5 cells with the NS5A-specific antibody E9E10 as described[11] and tissue culture infectious dose 50 (TCID50) was calculated.[28] Determination of Renilla luciferase was performed as described before.[29]

Quantification of HCV RNA and Core Protein

Quantification of HCV RNA was performed as described previously.[30] HCV core protein content in cell culture supernatants was measured with a commercially available diagnostic kit (Architect Anti-HCV, Abbott).

Western Blot Analysis

Immunoblot analysis was performed as described elsewhere.[26] Primary antibodies against core (C7-50), E2 (AP33), NS2 (6H6), and actin were used in a 1:1,000 dilution, except for the E2-specific antibody which was diluted 1:500.

Separation of Trans-Complemented HCV Particles on Density Gradient

Trans-complemented HCV particles were analyzed by iodixanol step gradient as described previously.[29]

Neutralization Assays

For quantification of antibody-mediated neutralization, inhibition levels of trans-complemented particle infection were analyzed by neutralization assay as described previously.[13, 30] Immunoglobulins were purified from sera of HCV patients infected with GT 1 (Ab1a_1 and Ab1a_2) or 2 (Ab2a_1) or healthy controls using the Thermo Scientific NAb Protein G Spin Kit. In addition, neutralization assays were performed with the monoclonal antibody AR4A and the control antibody RO4.

Results

Construction of a Jc1-Based Packaging Cell Line for Incorporation of Various Glycoproteins Into Single-Round Infectious HCV Particles

The HCV glycoproteins E1 and E2 are critically involved in virus assembly and cell entry. Retroviral pseudotypes carrying various E1-E2 proteins from cloned HCV consensus genomes or primary patient-derived isolates in place of retroviral envelope proteins (HCVpp) have become a popular model to investigate HCV cell entry.[9, 10, 31] However, HCVpp are produced in human nonliver cells and therefore lack a lipoprotein coat typical for authentic, cell culture-derived HCV particles (HCVcc) originating from human liver cells. Moreover, there are fundamental differences between cell entry of HCVpp and authentic HCVcc as, for instance, the Niemann Pick C1-like 1 cholesterol receptor (NPC1L1) is critical for HCVcc infection in vitro and in vivo but dispensable for HCVpp infection.[32] On the other hand, currently available HCVcc systems do not permit functional testing of multiple E1-E2 genes and therefore are not suited for in-depth analysis of functional traits of patient-derived envelope protein genes. To overcome these limitations we constructed an HCV packaging cell line constitutively expressing core, p7, and NS2 of the highly infectious GT 2a chimera Jc1 and confirmed protein expression by western blot[19] (Fig. 1A). Once these cells are transfected with a subgenomic JFH1 replicon expressing E1-E2 genes in the first cistron, all viral factors are available for production of infectious viral progeny. Notably, the resulting particles which are formed by way of trans-complementation (HCV trans-complemented particles, HCVTCP[23]), incorporate the replicon which lacks the coding regions for core, p7, and NS2. As a consequence, these HCVTCP are infectious for only a single round since the aforementioned critical viral assembly cofactors are not transduced to the infected cells, thus precluding further viral spread.

Figure 1.

Construction of a Jc1-based packaging cell line for incorporation of various E1-E2 proteins into single-round infectious HCV particles (HCVTCP). (A) Schematic representation of the Jc1 genome and the bicistronic JFH1 replicon expressing E1-E2 genes in the first cistron. Viral sequences derived from the J6 and JFH1 strains are depicted in dark and light gray, respectively and poliovirus-IRES (P-I) and encephalomyocarditisvirus-IRES (E-I) are given as black bars. Gene cassettes inserted into the packaging cell line by lentiviral gene transfer are indicated above the Jc1 genome. Expression of core and NS2 proteins in the packaging cell line was monitored by western blot. Note that the packaging cell line does not express functional E1-E2 genes. These are introduced by transfection of replicons encoding E1-E2 genes derived from given molecular clones of HCV[20] which represent all HCV genotypes. A replicon expressing the gene encoding gaussia luciferase (G-Luc) was used as negative control for virus production. E2 and NS5A protein expression at 48 hours posttransfection was monitored by western blot (B). Release of core protein at this timepoint was determined by a core-specific ELISA (C), while production of infectious progeny was determined 24, 48 and 72 hours posttransfection by using a limiting dilution assay (D). Mean values with standard error for particle production and core release are given from three and two independent experiments, respectively. Background level of the assay is shown in a dotted line.

To test if this cell line allows production of infectious HCVTCP with glycoproteins from all known HCV genotypes, we cloned replicons encoding the E1-E2 genes of representative isolates from each viral genotype and transfected these into the packaging cell line. As expected, all replicons propagated efficiently in the transfected cell, as evidenced by the accumulation of abundant NS5A protein (Fig. 1B). Different levels of E2 protein were detected, which likely reflects variation of the epitope targeted by the AP33 monoclonal antibody used for protein analysis. In parallel, we determined the release of HCV core protein into the culture fluid as marker for export of HCV particles (Fig. 1C). Notably, a modest level of core protein was detected upon transfection of the control replicon encoding a gaussia luciferase protein (G-Luc) instead of E1-E2 (Fig. 1C). However, these core structures released in the absence of viral glycoproteins were noninfectious, as evidenced by the limiting dilution assay displayed in Fig. 1D. In contrast, when replicons encoding GT 1 through GT 7-derived E1-E2 proteins were transfected, infectious progeny was produced except for the ED43 strain (GT 4a). J6 (GT 2a), JFH1 (GT 2a), and QC69 (GT 7a) E1-E2 proteins yielded peak infectious titers above 105 TCID50/mL correlating with maximal release of core protein (Fig. 1C,D). However, GT 1-derived E1-E2 proteins supported only modest virus production, with peak virus titers more than 1,000-fold lower. Similarly, virus production in the presence of J8 (GT 2b), S52 (GT 3a), SA13 (GT 5a), and HK6a (GT 6a) was comparatively low (Fig. 1D). Since all E1-E2 genes tested in the Jc1 packaging cell line are derived from viral consensus genomes proven to produce infectious HCV in cell culture and for some also in vivo, we can rule out that dysfunctional E1-E2 proteins limited virus production.[14] Notably, using chimeric HCV full-length genomes expressing E1-E2 genes from different viral genotypes we have reported that HCV envelope proteins support virus production in a genotype-dependent fashion.[33] This is likely because E1-E2 proteins interact with other viral proteins in a genotype-specific manner.[33] Therefore, incompatibility of E1-E2 proteins with the remaining viral factors in the context of the GT 2a Jc1-packaging cell line is likely due to limited virus production from replicons expressing E1-E2 sequences highly divergent from the cognate J6-derived E1-E2 proteins.

Nevertheless, a modest level of infectious progeny was produced for most tested E1-E2 genes and highly efficient virus release was observed for both GT 2a strains (JFH1 and J6) as well as a GT 7a isolate (QC69).

Efficient Production of Infectious Particles With Primary GT 2-Infected Patient-Derived Glycoproteins

These findings raised the hope that at least GT 2a-derived E1-E2 proteins may be readily incorporated onto infectious progeny in the background of the Jc1 packaging cell line. To test this, E1-E2 sequences from patient's plasma were obtained by RT-PCR, cloned into the bicistronic JFH1 helper replicon (Fig. 1A) and subsequently transfected into the Jc1 packaging cell line. Remarkably, four of five GT 2b-derived primary E1-E2 genes produced infectious progeny with peak titers ranging between 103 to 2 × 104 TCID50/mL (Fig. 2C). Moreover, three of five tested GT 2a-E1-E2 sequences yielded infectious virus between 2 × 104 to 2 × 105 TCID50/mL (Fig. 2C). A representative immunofluorescence picture of infection efficiency with H2a-4 and J6-derived glycoproteins is shown in Supporting Fig. S1. For those three sequences that did not produce detectable infectious virus (H2b-3, H2a-1, and H2a-5), we observed intracellular E2 protein expression (Fig. 2A) and at least for two of them increased liberation of HCV core protein, suggesting that for these two, primarily noninfectious viruses were released (Fig. 2B). In contrast, core release upon transfection of H2a-1 was similar to the G-Luc control, suggesting that for this strain release of virus was not supported. If failure to produce infectious virus in these three strains is due to amplification of nonfunctional E1-E2 sequences circulating in vivo or due to introduction of mutations by PCR is unknown. Nevertheless, our results show that the Jc1 packaging cell line permits efficient incorporation of primary GT 2a- and GT 2b-derived E1-E2 protein sequences onto single-round infectious HCVTCP particles for the majority of primary isolates (70%), rendering this model a powerful system to explore functional traits of primary GT 2-derived E1-E2 proteins in cell culture.

Figure 2.

Efficient production of infectious particles with genotype 2 patient-derived glycoproteins. JFH1-based replicons encoding given patient-derived E1-E2 genes were transfected into the Jc1-based packaging cell line lacking endogenous E1-E2 expression. A replicon encoding the J6-E1-E2 proteins or the gene encoding gaussia luciferase (G-Luc) was transfected as positive or negative control, respectively. E2 and NS5A expression were monitored by western blot (A) and core release was quantified by a core-specific ELISA (B). Production of infectious progeny was determined by a limiting dilution assay (C). Mean values with standard error for particle production and core release are given from three and two independent experiments, respectively. Background level of the assay is shown in a dotted line.

Construction and Characterization of an E1-E2 Packaging System for GT 1 Patient-Derived Glycoproteins

As depicted in Fig. 1D, the trans-complementation efficiency of GT 1 glycoproteins was low with viral titers of about 102 TCID50/mL in the Jc1-based packaging system. To overcome this limitation and to also incorporate GT 1-derived patient isolates in infectious particles, we aimed to develop a packaging system optimized for GT 1 E1-E2 sequences. Since virus production of H77 (GT 1a)-JFH1 full-length chimeric genomes was most efficient with a junction between H77 and JFH1 after residue 842 of the H77 polyprotein directly downstream of the first transmembrane domain of NS2 was chosen, our GT 1-packaging cells were constructed with this optimized junction for intergenotypic chimeras.[19] Moreover, long-term passage of H77-JFH1 full-length viruses has led to identification of adaptive mutations that increase virus production from these viruses. These are located within p7[34] (H77 V787A), and within NS3[34] (H77 Q1247L). Finally, Kaul et al.[35] reported that the V2440L exchange within the NS5A protein of JFH1 facilitates virus release of several intergenotypic JFH1 chimeras. To find out which adaptive mutations would permit optimal virus production for primary GT 1 E1-E2 proteins, we created two distinct packaging cell lines expressing H77 core, p7 with or without the adaptive mutation in p7 (Huh-7.5[C][p7NS2]H77/V787A and Huh-7.5[C][p7NS2]H77, respectively) and a chimeric H77/JFH1 NS2 protein (Fig. 3A). Moreover, three distinct replicons were constructed which all expressed the H77 E1-E2 proteins in the first cistron but encoded either a wild-type JFH1 replicase (NS3-NS5B), a replicase with the NS3 Q1247L mutation (NS3-NS5B/Q1247L), or a replicase with both the NS3 and the NS5A mutation (NS3-NS5B/Q1247l+V2440L). Subsequently, viral protein expression in the two different packaging cell lines was confirmed by western blot (Fig. 3B) and the efficiency of virus production upon transfection of these replicons was assessed by use of the limiting dilution assay (Fig. 3C). Notably, transfection of the wild-type replicon with H77 E1-E2 proteins into the packaging cell line expressing wild-type p7 yielded only a low virus titer (Fig. 3C, lane 1). In contrast, when the Q1247L mutation was encoded by the replicon (Fig. 3C, lane 3), ∼30-fold higher virus titers reaching up to 1 × 103 TCID50/mL were attained in both packaging cell lines (with or without p7 mutation; lanes 3 and 5, respectively), indicating that the Q1247L mutation is critical for production of HCVTCP with H77 envelope proteins. In line with this notion, use of the replicon encoding both the NS3 and NS5A mutations did not further improve virus production (Fig. 3C, lane 8). Thus, in conclusion, at least for H77, the aforementioned adaptive mutation in NS3 strongly increases assembly of infectious HCVTCP production in H77-JFH1 packaging cells.

Figure 3.

Construction and characterization of E1-E2 protein packaging systems for GT 1 patient-derived glycoproteins. (A) Schematic representation of the chimeric H77/C3 genome[19] and the bicistronic JFH1 replicon are given at the top. H77-derived sequences are depicted in white and JFH1-derived sequences in light gray. Adaptive mutations are indicated by asterisks. H77 E1-E2-encoding replicons with wild-type JFH1 sequence or with the Q1247L mutation, the V2440L mutation or with both adaptive changes were transfected into the packaging cell lines encoding either wild-type H77 genes (Huh-7.5[C]p7NS2]H77) or V787A adapted H77 genes (Huh-7.5[C]p7NS2]H77/V787A). H77 transgene expression in the packaging cell lines upon lentiviral gene transfer was monitored by western blot (B). Production of infectious progeny was monitored using the limiting dilution assay (C). Presence of adaptive mutations during virus production is indicated below each lane. Mean values with standard error are given from three independent experiments. Background level of the assay is shown in a dotted line.

To explore if these mutations also facilitate production of infectious HCVTCP particles carrying primary E1-E2 genes from GT 1a infected individuals we cloned six distinct E1-E2 sequences from GT 1a-infected patients and one from a GT 1b patient into JFH1-based helper replicons. Note that we created E1-E2-encoding replicons without adaptive mutations and with the Q1247L and V2440L mutations to explore the impact of adaptive mutations on incorporation of GT 1-derived E1-E2 proteins onto HCVTCP. Subsequently, wild-type replicons were transfected into the Jc1-based packaging cell line (Fig. 4A) or into the wild-type H77-packaging cell line (Fig. 4B), whereas the adapted replicon was transfected into the adapted H77-packaging cell line (Fig. 4C). Protein expression as well as virus production was monitored as described above. Irrespective of which cell line was transfected and which replicon context was chosen, we observed similar expression of E1-E2 protein genes and NS5A protein, suggesting that the cellular and the replicon background neither grossly affected E1-E2 protein expression nor replicon propagation (Fig. 4, upper panel). Notably, detection of E2 proteins was variable between patient isolates, which likely reflects variability of the epitope recognized by the AP33 antibody used for protein detection. Interestingly, release of core protein observed upon transfection of each E1-E2 encoding adapted replicon into the adapted H77 packaging cell line was clearly higher when compared to the G-Luc-encoding control replicon, suggesting that each of these replicons supported production and release of HCVTCP (Fig. 4C, middle panel). Moreover, in each case core release was higher compared to when the respective wild-type replicon was transfected into wild-type H77 packaging cells, suggesting that the adaptive changes facilitated virus production for all tested GT 1-derived E1-E2 sequences (Fig. 4, middle panel). Most important, production of infectious progeny was achieved for 8 of 10 tested GT 1-derived E1-E2 sequences in the adapted GT 1a packaging system and was between 10- to 100-fold higher compared to the wild-type H77 packaging and the Jc1-packaging system (Fig. 4, lower panel). A representative immunofluorescence picture of infection efficiency with H1a-6 and H77-derived glycoproteins is shown in Supporting Fig. S1.

Figure 4.

GT 1a-specific packaging system with adaptive mutations facilitates virus production with GT 1-patient-derived E1-E2 glycoproteins. (A) Wild-type JFH1-derived replicons expressing given E1-E2 sequences or gaussia luciferase were transfected into the Jc1 packaging cell line. Viral protein expression was determined by western blot and ELISA assay (upper and middle panel, respectively), whereas virus production 72 hours posttransfection was measured by a limiting dilution assay (lower panel). Alternatively, given wild-type JFH1 replicons were transfected into the GT 1a wild-type packaging cell line (B) or adapted JFH1 replicons were transfected into adapted GT 1a packaging cells (C). In both cases protein detection and virus production was monitored as described in (A). Mean values with standard error for particle production and core release are given from three and two independent experiments, respectively. Background level of the assay is shown in a dotted line.

Thus, in conclusion, by creating a custom-made H77-JFH1-based packaging system including a few adaptive mutations we were able to develop a robust cell culture system for production of authentic HCV particles carrying functional GT 1 patient-derived E1-E2 sequences.

Phylogenetic Analysis of HCV Glycoproteins for Productive Trans-Complementation

E1E2 sequences derived from standard laboratory strains representing genotypes 1-7 were aligned with patient-derived E1E2 glycoprotein sequences and used to generate a phylogenetic reconstruction using the best-fit nucleotide substitution model (GTR+I+Γ) under the maximum likelihood criterion (Fig. 5). As expected, patient-derived E1E2s clustered with reference sequences according to the viral genotype and therefore correlated with the observed genotype- and isolate-dependent efficiency in trans-complementation. Of note, the positioning of GT 7 in the E1E2 phylogeny indicates it is genetically more closely related to GT 2 than all other genotypes (Fig. 5).

Figure 5.

Positioning of patient-derived isolates within the E1E2 phylogeny. Unrooted radial tree for HCV E1E2 is depicted. Bootstrap values assigned to internal nodes indicate well-supported branches leading to the major HCV genotypes/subtypes. Isolate names are given at terminal nodes and genotype 1 and 2 sequences are highlighted in gray. E1E2 sequences, which produced functional virions in GT 1 or GT 2 trans-complementation assays, are denoted by ° or *, respectively. Branch lengths are relative to the scale bar and are proportional to nucleotide substitutions per site.

HCV Particles Bearing Patient-Derived Glycoproteins Have Authentic Buoyant Densities and Enter Target Cells in a CD81- and ApoE-Dependent Manner

To firmly establish that infectious particles carrying primary, patient-derived E1-E2 genes are authentic, we compared CD81- and ApoE-dependence of cell entry as well as buoyant density of released particles between H77, J6 laboratory strain-derived and H1a-6 and H2a-4 patient-derived HCVTCP particles (Figs. 6, 7). As depicted in Fig. 6A,B, anti-CD81 antibodies reduced infectivity of all tested HCVTCP particles to a similar degree. Moreover, ApoE-specific antibodies partially neutralized infection of all four tested HCVTCP types (Fig. 6C,D). Finally, these HCVTCP particles all shared the broad distribution of viral RNA, core, and infectivity including low density fractions that is typical for HCVcc particles and reflects their association with human lipoproteins (Fig. 7). In summary, these results show that HCVTCP carrying primary, patient-derived glycoproteins possess similar properties as authentic HCV infectious virions, including use of CD81 for target cell entry, close association with ApoE, and comparable buoyant density profiles.

Figure 6.

HCV particles carrying primary patient-derived E1-E2 proteins enter target cells in a CD81-dependent manner and are neutralized by ApoE-specific antibodies. (A,B) HCVTCP harboring primary GT 1a- (A) or GT 2a-derived glycoproteins (B) were incubated with CD81-antibodies for 1 hour at 37°C before inoculation of Huh-7.5 for 4 hours at 37°C. Infectivity was determined by focus forming unit assay. Mean values with standard error are given from three independent experiments. (C,D) Experimental procedure as above in the presence of ApoE antibodies.

Figure 7.

Density gradient analysis of HCV particles carrying primary patient-derived E1-E2 proteins. Given HCVTCP with either GT 1a (H77 and H1a-6) or GT 2a (J6 and H2a-4) E1-E2 sequences were harvested 72 hours after transfection of Huh-7.5 packaging cells and were resolved using an iodixanol step gradient. Nine to 11 fractions were harvested from bottom to top and HCV infectivity (A,D), core protein levels (B,E), and RNA copy numbers (C,F) were determined for each fraction. Values are plotted against the density of the respective fraction measured by refractometry. One representative experiment out of three independent experiments is shown.

Neutralization of Luciferase-Based Reporter HCVTCP With Patient-Derived Immunoglobulins and Monoclonal Antibodies

To facilitate the detection of infection events, we next generated reporter replicons for incorporation of patient-derived GT 1- and GT 2- E1-E2 genes into reporter virus particles (Supporting Fig. S2A). To this end, we fused a Renilla luciferase gene to the N-terminus of the NS3-NS5B coding region by way of a foot-and-mouth disease virus 2A peptide sequence so that the reporter is liberated from the HCV polyprotein after translation. These novel reporter genomes replicated to high levels in transfected Huh-7.5.1-based GT 2a (Supporting Fig. S2B) and GT 1a (Supporting Fig. S2C) packaging cell lines, which were generated to enhance the efficiency of trans-complementation. Moreover, production of infectious particles was determined by a highly sensitive luciferase assay and was more efficient in the GT 2a packaging system compared to the GT 1a adapted packaging system, as observed before (Supporting Fig. S2D,E). Next, we analyzed the neutralization susceptibility of HCVTCP incorporating GT 2a (Fig. 8A-D) and GT 1a-derived glycoproteins (Fig. 8E,F) to antibodies derived from genotype-matched patient sera and in case of GT 2a glycoproteins also to the monoclonal antibody AR4A which broadly neutralizes several HCV genotypes.[36] Purified immunoglobulins from an HCV GT 2-infected patient (Ab2a_1) neutralized J6- as well as GT 2a-patient derived glycoproteins in a dose-dependent manner (Fig. 8A). Moreover, the AR4A monoclonal antibody inhibited infection of GT 2a reporter viruses, with the H2a-4-derived glycoproteins being somewhat more sensitive to neutralization by this antibody compared to the J6-derived envelope proteins (Fig. 8C). In the same line, GT 1a-derived glycoproteins were neutralized by purified antibodies from GT 1-infected patients (Ab1a_1 and Ab1a_2) with slight differences between H77- and patient isolate-based trans-complemented particles (Fig. 8E,F), with no cytotoxicity observed (data not shown). In conclusion, primary patient-derived glycoproteins incorporated into infectious particles can be neutralized by patient-derived immunoglobulins and monoclonal antibodies and analyzed by a highly sensitive reporter assay.

Figure 8.

HCV reporter particles carrying primary patient-derived E1-E2 proteins are neutralized by immunoglobulins from patient sera. (A-G) Luciferase reporter-based HCVTCP harboring primary GT 2a- (A-D) or GT 1a-derived glycoproteins (E,F) were preincubated for 1 hour at 37°C with antibodies purified from HCV-infected patients (Ab2a_1, Ab1a_1, and Ab1a_2) or the monoclonal antibody AR4A. Immunoglobulins from healthy donors or the RO4 monoclonal antibody, recognizing a cytomegalovirus protein, served as controls. Huh-7.5 cells were inoculated for 4 hours at 37°C and infectivity was determined by Renilla luciferase assays after 72 hours. Mean values with standard error are given from at least two independent experiments.

Discussion

By exploiting the ability of HCV to incorporate subgenomic replicons into single-round infectious particles, in this study we developed cell culture systems for production of authentic, infectious HCV incorporating primary patient-derived glycoproteins from GT 1- and GT 2-infected individuals. Due to expression of E1-E2 genes from a single gene cassette inserted into autonomously replicating HCV replicons, potential issues resulting from aberrant polyprotein processing are excluded and simple cloning of a large number of patient-derived E1-E2 genes is facilitated. We feel that these novel systems encompassing also reporter-based infection readouts are attractive models for studying E1-E2 protein function in an individualized fashion.

Importantly, several lines of evidence provided in this study highlight that these particles recapitulate key features of authentic E1-E2 protein function both in terms of virus assembly and cell entry. Specifically, we show that these particles infect Huh-7.5 cells in a CD81-dependent fashion and can be neutralized by patient-derived antibodies or monoclonal antibodies. Moreover, neutralization of infection by ApoE-specific antibodies as well as the heterogeneous density distribution of cell-free HCVTCP indicate that these viruses incorporate host-derived lipoproteins. This latter finding is particularly relevant since HCVpp, which have been used previously for assessment of primary patient-derived glycoprotein function, are produced in a human kidney cell line (293T) that lacks endogenous expression of human lipoproteins, thus confounding the usefulness of HCVpp for analysis of the interplay between E1-E2 proteins with lipoproteins. Moreover, HCVpp do not permit studies of glycoprotein functions during virus assembly since these particles are assembled in the absence of other HCV factors and lipoproteins. In contrast, HCVTCP, like full-length HCV, require expression of all viral structural and nonstructural proteins as well as apolipoproteins for release of infectious progeny.[23, 37] Moreover, we show that E1-E2 genes are incorporated into HCVTCP in an isolate- and genotype-dependent fashion. This finding is consistent with our previous observation with chimeric full-length genomes carrying E1-E2 genes from isolates deviating from the other viral genes.[33] In this previous work we could show that genetic incompatibility of E1-E2 proteins with remaining viral factors during assembly limits envelopment of HCV nucleocapsids likely due to inefficient protein-protein interactions between the glycoprotein complex and other viral proteins.[33] The results displayed in this present study show that genetic compatibility of E1-E2 also governs production of HCVTCP confirming that this system recapitulates authentic viral assembly pathways.

Clearly, genetic incompatibility of glycoproteins with viral factors derived from other HCV genomes restricts use of a single packaging cell line for incorporation of E1-E2 proteins across all viral genotypes. Nevertheless, our data indicate that within a given HCV genotype insertion of E1-E2 genes from various isolates is well tolerated, as 70 to 80% of tested GT 2 and GT 1 isolates produced robust infectious titers in their cognate packaging systems. Notably, the packaging system based on the Jc1 chimera,[19] which grows to very high titers in cell culture,[33] is particularly suited to produce highly infectious HCVTCP. This is likely attributable to specific features of J6-derived core and p7 protein functions supporting a high level of virus production.[33] However, these beneficial properties of the J6 strain do not overrule the requirement for intragenotypic compatibility of the E1-E2 sequences since the GT 1a packaging system with adaptive mutation rescues a greater proportion of GT 1-derived E1-E2 isolates (80% instead of 70% for the Jc1 packaging system) and consistently yields higher infectious titers. Therefore, construction of custom-made packaging systems for each HCV genotype should be a reasonable strategy to ultimately create cell culture systems that permit assessment of functional E1-E2 protein traits across all naturally occurring HCV strains. The infectious full-length JFH1-based chimeric HCV genomes including distinct adaptive changes should be a reasonable starting point for these efforts.[20]

Ultimately, in-depth functional comparison between HCV E1-E2 protein sequences should shed further light on the requirements for E1-E2 function and protein-protein interactions during virus assembly. Moreover, these experiments may reveal unique features of the interplay between E1-E2 and lipoproteins and host cell entry factors. Finally, these models should be instrumental for exploring viral resistance to HCV entry inhibitors and ultimately for development of vaccine candidates that elicit broadly protective cross-neutralizing antibodies.

Acknowledgment

We thank Takaji Wakita (National Institute of Infectious Diseases, Tokyo), Jens Bukh and Judith Gottwein (Copenhagen University Hospital, Copenhagen) for JFH1 and infectious chimeric HCV constructs, respectively; Charles Rice (Rockefeller University, New York City) for Huh-7.5 cells, 6H6, and E9E10 monoclonal antibody; Darius Moradpour (Centre Hospitalier Universitaire Vaudois, Lausanne) for the core-specific C7-50 antibody and to GENENTECH; Arvind Patel (University of Glasgow, Glasgow) for providing AP33; Frank Chisari (Scripps Research Institute, La Jolla, CA) for Huh-7.5.1 cells; Mansun Law (Scripps Research Institute, La Jolla, CA) for the AR4A antibody; Steven Foung (Stanford University School of Medicine, Stanford, CA) for the RO4 antibody; Francois Penin (Institut de Biologie et Chimie des Proteins) for helpful discussion; and all members of the Institute of Experimental Virology, Twincore, for helpful suggestions and discussions.

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