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Previous Article | Next Article 
Journal of Virology, May 2007, p. 4591-4603, Vol. 81, No. 9
0022-538X/07/$08.00+0 doi:10.1128/JVI.02144-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Nicole Appel,
George Koutsoudakis,
Stephanie Kallis,
Volker Lohmann,
Thomas Pietschmann, and
Ralf Bartenschlager*
Department for Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
Received 29 September 2006/ Accepted 4 February 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HCV genome is an uncapped linear single-stranded RNA molecule of positive polarity and has a size of 
9.6 kb. The RNA encodes a large polyprotein of about 3,000 amino acids in a single open reading frame which is flanked at the 5' and 3' ends by nontranslated regions (NTRs). Both the 5' and 3' NTRs are required for RNA translation and replication (reviewed in reference 4). Expression of the polyprotein is initiated at an internal ribosome entry site (IRES) located in the 5' NTR. The polyprotein is processed co- and posttranslationally by cellular and viral proteases into the structural proteins (core, E1, and E2) and the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). In addition, the production of a novel HCV protein generated by internal translation initiation or by ribosomal frameshifting has been reported, but its function remains unclear (reviewed in reference 8). The nonstructural proteins NS3 to NS5B are sufficient for RNA replication (35), and distinct functions in the HCV life cycle have been described for most of them. However, the functional role of the phosphoprotein NS5A is still enigmatic. Based on tryptic digestion patterns and biochemical as well as structural analyses, NS5A is composed of three domains separated by low-complexity regions (46) (Fig. 1). In addition, an amphipathic 
-helix resides at the very N terminus of the protein and mediates NS5A membrane attachment (9). Recently, the crystal structure of NS5A domain 1 was solved, and it was suggested that NS5A interacts with viral RNA (21, 41, 47). Interestingly, NS5A domain 3 is only poorly conserved between different genotypes and tolerates deletions as well as insertion of large heterologous sequences, such as green fluorescent protein (GFP), which allows the visualization of replication in living cells (2, 38).
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Several reports have shown that a cell infected with a virus often becomes resistant to secondary infection with the same (homologous) virus, whereas infection with nonrelated (heterologous) viruses is frequently unaffected. This phenomenon, called superinfection exclusion, is brought about by several mechanisms acting at various stages of the viral life cycle. These include interference at the level of virus entry, inhibition of translation, and interference with replication of the incoming RNA (1, 12, 18, 22, 23, 32, 51). For HCV, it is not clear whether superinfection exclusion is induced upon primary infection.
In this study, we developed a panel of HCV genomes that allow direct visualization of infected cells, using fluorescence microscopy, and discrimination between viral genomes in infected cells. By utilizing this system to study superinfection exclusion, we found that preceding infection with HCV strongly interferes with secondary HCV infection. This block is exerted already 24 h after primary infection, and it is mainly due to interference at the level of RNA translation/replication, whereas viral entry is largely unaffected.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid construction.
All nucleotide and amino acid numbers refer to the JFH-1 genome. Construction of the Jc1 chimera was described recently (42). To generate fluorochrome gene-tagged reporter plasmids, we performed two separate PCRs, using antisense primer Alinker_Xba_Pme_NS5A_aa383 (5'-TTAAACCCAGGTCTAGAACCGCTCGAGGGGGGCTGGCCAAAG-3') and sense primer SRsrII (5'-CCGAATCCGGCGGTCCGACGTCCC-3') or sense primer Slinker_Xba_Pme_NS5A_aa383 (5'-TCTAGACCTGGGTTTAAACGTGATGCAGGCTCGTCCACGGG-3') and antisense primer AHpaI (5'-GACTTGATGTGGTTAACGGCCCTC-3') for insertion of the linker at NS5A amino acid position 383 and antisense primer Alinker_Xba_Pme_NS5A_aa383 (5'-TTAAACCCAGGTCTAGAACCGCTCGAGGGGGGCTGGCCAAAG-3') and sense primer SRsrII or sense primer Slinker_Xba_Pme_NS5A_aa383 (5'-TCTAGACCTGGGTTTAAACGTGATGCAGGCTCGTCCACGGG-3') and antisense primer AHpaI for insertion at NS5A amino acid position 378. Amplicons were combined by a second PCR resulting in fragments that comprise the complete NS5A coding region plus the linker, which were HpaI/RsrII digested and transferred into pFKi389LucNS3-3'_dg_JFH. This plasmid contains the T7 promoter sequence fused to nucleotides 1 to 389 of the JFH-1 consensus sequence, followed by the firefly luciferase gene, which is flanked by in-frame AscI and NotI restriction sites; the encephalomyocarditis virus (EMCV) IRES; the NS3-to-NS5B coding sequence; the 3' NTR of JFH-1; the hepatitis delta virus genomic ribozyme (dg); and the T7 terminator sequence (Lohmann, unpublished data). Venus-GFP (39), kindly provided by C. M. Brown, University of Otago, Dunedin, New Zealand, emerald GFP, and RFP reporter genes were amplified by PCR and inserted, after restriction digestion with XbaI and PmeI, into the linker plasmids to create reporter plasmids pFKi389LucNS3-3'_dg_JFH-1_NS5Aaa2354_Venus, pFKi389LucNS3-3'_dg_JFH-1_NS5Aaa2359_emGFP, pFKi389LucNS3-3'_dg_JFH-1_NS5Aaa2354_RFP, and pFKi389LucNS3-3'_dg_JFH-1_NS5Aaa2359_RFP, respectively. To generate NS5A fluorochrome gene-tagged Jc1 genomes, an NsiI/MluI fragment including the NS5A coding region was transferred from subgenomic replicon plasmids to the pFK-J6/C3 chimera carrying the Jc1 virus sequence (42). The construct Venus-Jc1 is described elsewhere (30) and represents a bicistronic reporter virus genome carrying the coding region of Venus-GFP in the first cistron and the chimeric Jc1 open reading frame in the second cistron, expressed by means of the EMCV IRES. Plasmid pFKi389neoNS3-3'_dg_JFH-1_NS5Aaa2359_RFP was used to generate sg/neo/5A-RFP and constructed by replacing the firefly luciferase gene in pFKi389LucNS3-3'_dg_JFH-1_NS5Aaa2359_RFP with the gene encoding neomycin phosphotransferase, using AscI and NotI restriction sites flanking the luciferase gene. All PCR-amplified DNA fragments were analyzed by automated nucleotide sequencing with an ABI 310 sequencer (Applied Biosystems). Big Dye Terminator, version 1.1 (Applied Biosystems), was used for cycle sequencing according to the manufacturer's protocol. The HCV E1E2 expression construct phCMV
cE1/E2-Con1 was derived from the phCMV-7a expression vector (5) by replacing the DNA fragment encoding the last 60 residues of HCV core and all of E1 and E2 of the genotype 1a H77 isolate with the corresponding fragment of the HCV genotype 1b isolate Con1 (GenBank accession number AJ238799) (35). The HCV E1E2 expression constructs pcDNA3cE1E2-JFH1 and pcDNA3cE1E2-J6CF were constructed in an analogous way, and they direct the expression of envelope glycoproteins of the HCV isolates JFH-1 (28) and J6CF (52), which both belong to genotype 2a.
In vitro transcription, electroporation of HCV RNAs, and Northern blot analysis. In vitro transcripts of the individual constructs were generated by linearizing the respective plasmids by digestion with MluI. In vitro transcription and electroporation of HCV RNAs were performed as described previously (29). For generation of capped transcripts encoding Renilla luciferase, the same protocol was applied except that the nucleotide mix contained 1 mM of the cap analog m7G(5')ppp(5')G (New England Biolabs), 1.25 mM GTP, a 2.5 mM concentration (each) of ATP, CTP, and UTP, and pRL CMV (Promega) as a template after linearization with BamHI. For Northern hybridization, total RNA was prepared by a single-step isolation method (10). After gel electrophoresis and membrane blotting, the membrane was incubated with a 32P-labeled negative-sense riboprobe complementary to nucleotides 6273 to 9678 of the JFH-1 genome as described previously (29). Hybridization with a ß-actin-specific antisense riboprobe served to correct for the total RNA amount loaded in each lane of the gel. Signals were detected and quantified by autoradiography using Kodak photofilms or phosphorimaging.
Immunofluorescence analysis. Cells were seeded onto glass coverslips that were placed in 24-well cell culture plates. At different time points after transfection or 72 h after infection, cells were washed three times with phosphate-buffered saline (PBS) and fixed with 500 µl of 4% paraformaldehyde for 10 min at room temperature (RT). Subsequently, cells were washed three times with PBS, permeabilized by a 5-min incubation in 500 µl of 0.5% Triton X-100 in PBS, and washed three times with PBS prior to incubation with the first antibody. The JFH-1 NS5A-specific antibody 9E10 (33), kindly provided by T. Tellinghuisen and C. M. Rice, Rockefeller University, NY, was diluted 1:200 in PBS containing 5% goat serum. After 45 min of incubation at RT, cells were washed three times with PBS for 10 min each and incubated with a 1:1,000 dilution of the Alexa 488-conjugated secondary antibody. After 45 min of incubation in the dark, cells were washed once with PBS, incubated for 1 min with a 1:5,000 diluted 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI)-PBS solution, and immediately washed three times for 10 min each with PBS. Finally, cells were washed with water and mounted on glass slides with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). For detection of dengue virus 2 (DEN-2) infection, an NS5-monospecific polyclonal antiserum raised in rabbits was used as described recently (36). Bound antibodies were detected by using a goat anti-rabbit Alexa 488-conjugated secondary antibody (Molecular Probes, Leiden, The Netherlands).
FACS analysis.
Huh7-Lunet cells were transfected with 10 µg in vitro transcript of Jc1/GFP, Venus-Jc1, JFH1E/GFP, or Jc1, seeded into six-well culture dishes, and detached by trypsin treatment at 24, 48, and 72 h posttransfection. Infected cells were grown in 12-well culture dishes and analyzed at 72 h postinfection. Cells were resuspended in complete DMEM and centrifuged for 5 min at 4°C at 110 x g in a tabletop centrifuge. The cell pellet was resuspended in 3% paraformaldehyde and stored at 4°C until use. Immediately before analysis, cells were centrifuged for 5 min at 4°C at 110 x g, the paraformaldehyde solution was aspirated, and cells were resuspended in PBS supplemented with 0.2% bovine serum albumin and 0.02% sodium azide (fluorescence-activated cell scanning [FACS] assay buffer) and analyzed directly by using a FACSscan apparatus and Cell Quest Pro software (Becton Dickinson Biosciences). In case of simultaneous determination of cell surface expression of HCV receptor CD81 or scavenger receptor BI (SR-BI) with GFP-derived autofluorescence, detached cells were stained for 1 h on ice with a CD81-specific (115-136-071; Dianova, Hamburg, Germany) or SR-BI-specific (anti-CLA1; Becton Dickinson Biosciences) monoclonal antibody diluted 1:200 or 1:50, respectively, in FACS assay buffer. Subsequently, cells were washed with PBS, and bound antibodies were detected by a 1-h incubation on ice with mouse-specific secondary antibodies conjugated to allophycocyanin (Dianova, Hamburg, Germany) at a dilution of 1:100 in FACS assay buffer. Cells were analyzed by using a FACSCalibur apparatus and Cell Quest Pro software (both from Becton Dickinson Biosciences).
Luciferase assays. Transient HCV RNA replication assays were performed as described previously (31). In brief, 4 x 106 cells were transfected with 5 to 10 µg in vitro-transcribed RNA and resuspended in 12 ml or 20 ml culture medium (in cases of subgenomic or genomic RNA, respectively), and 2-ml aliquots were seeded into each well of a six-well culture dish. At 4 to 72 h posttransfection, cells were lysed and luciferase activity was determined. Renilla luciferase activity was measured in the same lysates, using an assay buffer containing 15 mM potassium phosphate (pH 7.8), 25 mM glycyl-glycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, and 50 µg/ml coelenterazine (PFK Chemikalien, Kleinblittersdorf, Germany).
Core ELISA. To quantify the amount of HCV core protein in transfected cells or cell culture supernatant, an Ortho Trak-C enzyme-linked immunosorbent assay (ELISA) kit was used (Ortho Clinical Diagnostics, Neckargemünd, Germany). Four million cells were transfected with 5 to 10 µg in vitro-transcribed RNA and resuspended in 20 ml culture medium, and 2-ml aliquots were seeded into each well of a six-well culture dish. Cell lysates and cell culture supernatants were analyzed at different time points between 4 h and 72 h posttransfection. Culture supernatants were used directly for core ELISA, whereas for determination of intracellular core amounts, cells were lysed by the addition of 500 µl PBS containing 1% Triton X-100, a 1/10,000 volume of aprotinin (1 U/ml), a 1/1,000 volume of leupeptin (4 mg/ml), and a 1/100 volume of phenylmethylsulfonyl fluoride (100 mM). After 5 min of centrifugation at 18,000 x g, supernatants were processed for ELISA according to the manufacturer's protocol. Colorimetric measurements were performed using a Sunrise colorimeter (Tecan Trading AG, Switzerland). To determine the efficiency of core protein release, the ratio of total core protein (the sum of intra- and extracellular core amounts) to extracellular core protein was determined.
TCID50 determination. Determination of 50% tissue culture infective dose (TCID50) values was performed as described recently, with slight modifications (33). Huh7.5 target cells were seeded at a concentration of 104 cells per well of a 96-well plate in a total volume of 180 µl complete DMEM. Twenty-four hours later, serial dilutions of virus-containing supernatant were added to eight wells per dilution. Virus-containing supernatant was replaced at 4 h postinoculation by fresh medium, and 72 h later, cells were washed with PBS and fixed by the addition of 100 µl ice-cold methanol and overnight incubation at –20°C. Cells were washed three times with PBS, permeabilized, and blocked overnight by the addition of 40 µl blocking solution (1x PBS, 0.5% saponin, 1% bovine serum albumin, 0.2% dried milk, 0.02% NaN3). Cells were then incubated with PBS-0.3% H2O2 (vol/vol) for 5 min at RT. After three washes with PBS and one wash with PBS containing 0.5% saponin, infected cells were detected by using the NS5A antibody 9E10 (1:200 dilution in PBS-0.5% saponin) and a goat anti-mouse secondary antibody coupled to horseradish peroxidase (A4416; Sigma). After a 1-h incubation at RT, the cells were washed three times with PBS and once with PBS-0.5% saponin. Horseradish peroxidase activity was detected by using a Vector Nova Red substrate kit (Linaris, Weinheim, Germany). Reactions were terminated by replacing the substrate solution with water. Plates were analyzed by microscopy, and virus titers were determined by using the calculation method of Spearman and Kärber (26, 45).
Generation of HCV pseudoparticles (HCVpp) and infection studies.
Human immunodeficiency virus (HIV)-based pseudotypes bearing vesicular stomatitis virus glycoproteins (VSV-G) or HCV Con1-, JFH-1-, or J6CF-derived E1 and E2 proteins were generated by cotransfection of 293T cells (14). Briefly, 2.5 x 106 293T cells were seeded into 10-cm-diameter plates 1 day before transfection with 2.7 µg envelope protein expression construct pczVSV-G (24), phCMVcE1-E2-Con1, pcDNA3cE1E2-JFH1, or pcDNA3cE1E2-J6C, 8.1 µg HIV Gag-Pol expression construct pCMVR8.74 (15), and 8.1 µg of a firefly luciferase transducing retroviral vector (29, 55) by using Lipofectamine 2000 (Invitrogen, Karslruhe, Germany). The medium was replaced 8 h after transfection, and supernatants containing the pseudoparticles were harvested 48 h later. Transfected cells were supplemented with fresh culture medium, and virus-containing cell culture fluid was harvested again 24 h later (at 72 h posttransfection). Both harvests were pooled, cleared by passage through 0.45-µm-pore-size filters, and concentrated by using Amicon ultrafiltration devices (Millipore). Concentrated (20-fold) pseudotype virus stocks were utilized to infect naïve Huh7.5 cells or Huh7.5 cells that had been infected with Jc1 virus 4 days earlier, using a multiplicity of infection (MOI) of 0.8. The efficiency of pseudotype virus infection was evaluated by luciferase assays at 72 h postinfection. HIV-based retroviral constructs were kindly provided by D. Trono, Swiss Institute of Technology, Lausanne, Switzerland.
DEN-2 and HCV coinfection analysis. Huh7.5 cells (1 x 105) were seeded onto glass coverslips and infected 24 h later with Jc1 virus at an MOI of 10. Cells were grown for 24 h and infected for 4 h with DEN-2 (New Guinea C strain) at an MOI of 2 as described recently (36). After 4 h, the inoculum was replaced by DMEM, and cells were incubated for another 24 h. Finally, cells were analyzed for HCV and DEN-2 infection by NS5A- and NS5-specific immunofluorescence, respectively.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E/GFP, lacking most of the envelope protein coding region, served as a negative control for particle production, and JFH1/GND, carrying an inactivated NS5B polymerase, served as a negative control for RNA replication. In addition, we used Venus-Jc1, a bicistronic full-length genome expressing the Venus variant of GFP (39) from the first cistron and the chimeric Jc1 open reading frame with unmodified NS5A from the second cistron (Fig. 1B) (30). This bicistronic construct design has been used in the past both for replicons and for a JFH-1 reporter virus (49) and therefore was used for a comparative analysis with the monocistronic constructs in order to determine the advantages and disadvantages of both systems.
In a first experiment, RNA replication in transfected cells was monitored by Northern hybridization (Fig. 1C). Replication of the monocistronic genomes Jc1/GFP and Jc1/RFP and of the JFH1E/GFP control was comparable to that of the Jc1 wild type, arguing that the insertion into NS5A had no influence on RNA replication capacity and kinetics. In contrast, we observed a clear delay in replication kinetics of the bicistronic genome Venus-Jc1. While replication of all monocistronic genomes had already increased significantly within the first 24 h posttransfection, the RNA amounts for the bicistronic genome Venus-Jc1 dropped below the input level during this time. Comparable results were obtained when transfected cells were examined by FACS analysis (data not shown). Moreover, we tested the analogous series of GFP constructs in the context of the authentic JFH-1 genome and found that replication of the monocistronic reporter genome JFH1/GFP was comparable to that of the JFH-1 wild type, while replication of the bicistronic Venus-JFH1 construct was lower and kinetics were delayed, similar to those of the Jc1 constructs (data not shown). Thus, the in-frame insertion into NS5A did not interfere with efficient RNA replication.
Production of infectious HCV particles from cells carrying fluorochrome gene-tagged HCV genomes.
To analyze particle production from cells transfected with the different reporter genomes, we measured the amounts of core protein released into cell culture supernatants in relation to total core protein amounts expressed in these cells. Supernatants and cells were harvested at 4, 24, 48, 72, and 96 h posttransfection and used for core-specific ELISA. As shown in Fig. 2A, comparable amounts of core protein were detected in cells 4 h after transfection, reflecting very similar transfection efficiencies. While core protein accumulated to comparable levels in cells transfected with the monocistronic constructs Jc1/GFP, Jc1/RFP, and JFH1E/GFP, core amounts obtained with the bicistronic Venus-Jc1 genome were reduced nearly 10-fold at 24 h posttransfection, most likely due to the delayed RNA replication kinetics. In contrast, the reduced intracellular core amounts in Jc1-transfected cells were probably the result of a very efficient core release already at 24 h posttransfection (Fig. 2B). Surprisingly, despite efficient RNA replication and intracellular core protein production, the relative amount of extracellular core protein released from cells that had been transfected with the monocistronic Jc1/GFP or Jc1/RFP genome was about 10-fold lower than that for the wild type (Fig. 2A, lower panel). In essence, in the case of Jc1, about 10% of total core protein was released into the supernatant 24 h after transfection (Fig. 2B). Core release achieved with the bicistronic Venus-Jc1 genome was up to 5% of the total core protein, while for the monocistronic genome <1% of the core protein was released from transfected cells. These results show that both the bicistronic and the monocistronic reporter genomes are impaired in virus production, but the defect is much more pronounced in the latter.
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Induction of superinfection exclusion by primary HCV infection. Having characterized the reporter gene-tagged viral genomes, we utilized them to find out whether primary HCV infection induces superinfection exclusion as described for several other viruses, including the closely related pestiviruses (32, 37). Huh7.5 cells were inoculated simultaneously with the monocistronic Jc1/GFP and Jc1/RFP viruses, with each at a multiplicity of infection (MOI) of 10 TCID50/cell. In parallel, we first infected cells only with Jc1/GFP and then, after 24 h or 48 h, performed secondary infection with Jc1/RFP (Fig. 4A). Seventy-two hours after the last infection, cells were fixed, and the number of GFP- or RFP-positive cells was determined by fluorescence microscopy. As shown in Fig. 4A and B, when cells were infected with Jc1/GFP and Jc1/RFP simultaneously, approximately 18% of all infected cells expressed both GFP and RFP, indicating that they had been infected by both viruses. Interestingly, in doubly infected cells, one virus species appeared to dominate over the other, as inferred from the inverse correlation of the intensities of GFP and RFP fluorescence. Very rarely, cells were detected in which GFP and RFP were expressed to the same level. In contrast, when Jc1/RFP infection was performed 24 h after primary infection with Jc1/GFP, only 2% of all infected cells displayed dual fluorescence, although >80% of the total cell population became infected, with 57% being infected with only Jc1/GFP and 41% being infected with only Jc1/RFP. After a 48-h primary infection with Jc1/GFP, the number of GFP-positive cells increased further, concomitant with a further reduction of RFP-positive cells and a constantly very small number of double-positive cells. These results suggest that primary HCV infection with Jc1/GFP induces a stringent level of superinfection exclusion within 24 h. The finding that even 48 h after primary infection a small number of cells can still be superinfected suggests that some HCV-infected cells may not be entirely refractory to a secondary infection or that these cells had been infected only poorly with the primary virus. Another possibility may be that double-positive cells are the result of a delayed (secondary) infection caused by progeny virus released from primary infected cells. In this case, secondary infection by Jc1/RFP occurred prior to the establishment of superinfection exclusion. However, this possibility is rather unlikely due to the high MOI chosen for primary infection with Jc1/GFP.
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Having shown that secondary infection of Huh7 cells with a homologous virus (HCV) was blocked, we wanted to exclude that this phenomenon was due to the induction of a general antiviral state as a result of primary infection. Therefore, Jc1-infected cells were subsequently infected with DEN-2. As shown in Fig. 4E, HCV-infected cells remained fully susceptible to infection with DEN-2, arguing for homologous interference being responsible for HCV superinfection exclusion rather than a general antiviral state.
No evidence for down regulation of CD81 and SR-BI on the surfaces of HCV-infected cells. Superinfection exclusion can be due to interference at the level of virus entry or RNA translation/replication. Superinfection exclusion can also occur at the level of virus assembly or release under conditions of nonsaturating primary infection where virus spread is still possible. To analyze whether HCV infection results in a down regulation of known molecules involved in HCV entry, we performed FACS analysis of Huh7.5 cells that had been infected with Venus-Jc1 for 72 h. We focused our analysis on CD81 and SR-BI, two molecules that are implicated in HCV entry. As presented in Fig. 5A, Venus-Jc1 productively infected about 26 to 30% of the total cell population. Importantly, the CD81- and SR-BI-specific mean fluorescence intensities in Venus-Jc1-infected cells did not significantly deviate from those in uninfected cells, indicating that for up to 72 h postinoculation, HCV infection did not affect CD81 and SR-BI densities on the cell surface. The same results were found with infection times of up to 120 h (not shown) and with higher MOIs (30).
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Superinfection exclusion is induced primarily at the level of RNA translation/replication. To study whether superinfection exclusion was due to a block at the level of RNA translation/replication, we transfected comparable amounts of a subgenomic reporter replicon containing the firefly luciferase gene in the first cistron and the GFP-tagged NS5A replicase gene in the second cistron into naive Huh7-Lunet cells or cells containing a subgenomic neo/5A-RFP replicon (Fig. 6A). The latter represents a cell pool obtained after transfection of Huh7-Lunet cells and G418 selection. In addition, cured subgenomic neo/5A-RFP replicon cells lacking detectable amounts of HCV (Fig. 6C) were used for comparison. They were generated by passaging of the cells in the presence of a selective inhibitor and served as a control to exclude nonspecific interference with the cells carrying the replicon. Transfected cells were harvested for a luciferase assay to monitor RNA replication (Fig. 6B) or fixed for fluorescence microscopy to study protein expression at the single-cell level (Fig. 6C). Luciferase activity detected 4 h after transfection was set to 1 relative light unit (Fig. 6B). At 24 h posttransfection and all time points thereafter, a 10-fold reduction of luciferase activity was observed with replicon cells compared to that for naive or cured cells, indicating that prior presence of the replicon interfered with replication of the incoming RNA.
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To discriminate whether interference occurred at the level of RNA translation or RNA replication, we cotransfected a replication-deficient subgenomic RNA, designated sg/luc/GDD, and a capped Renilla luciferase RNA into subgenomic neo/5A-RFP replicon cells and the corresponding cured cells (Fig. 7). Cells were harvested 2, 4, 6, and 9.5 h after transfection, and luciferase activity was determined. Renilla luciferase activities achieved by cap-dependent translation were similar for both cell pools. In contrast, HCV IRES-dependent translation measured by firefly luciferase activity was reduced nearly twofold in replicon-containing cells compared to that in cured cells. These results raise evidence that part of the superinfection exclusion occurs at the level of RNA translation. In summary, these results suggest that superinfection exclusion is due, at least in part, to interference at the level of RNA translation, but given the moderate reduction, it is likely that exclusion also occurs at the level of RNA replication.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In agreement with an earlier report (29), we found that the RNA replication kinetics of the bicistronic reporter virus genome (Venus-Jc1) was delayed 24 h compared to that of the wild type, concomitant with a delayed release of infectious virus particles. Interestingly, RNA replication of the monocistronic reporter genomes was unaffected, but release of infectious virus particles was significantly reduced. In fact, we observed an about 50-fold lower infectivity titer than that of the Jc1 wild type, which most likely was due to an impairment of virus particle production rather than an impairment of virus infectivity. Currently, it is not clear whether particle assembly or particle release is affected. Alternatively, the insertion into domain 3 of NS5A may indirectly affect the functions of other viral or cellular proteins responsible for virus particle production. Further studies are required to identify a possible role for NS5A in virus production.
One possibility for achieving higher virus titers while retaining the reporter gene is to use bicistronic genomes. However, there are several disadvantages inherent to this construct design. First, presumably due to their increased length or the presence of an internal highly structured RNA that may slow down RNA synthesis, replication of these genomes is significantly delayed compared to that of monocistronic genomes. Second, during prolonged passage we observed that expression of the GFP reporter gene was rapidly lost, most likely due to deletion of all or most of the heterologous sequences. Since the mutants have much faster replication kinetics, they rapidly become the predominant virus species. In contrast, replication of the monocistronic reporter viruses is similar to that of the Jc1 wild type. Moreover, deletion mutants in which most or all of the heterologous sequences have been removed are only viable in case of an in-frame deletion, which is less likely in monocistronic reporter viruses than in the bicistronic virus genome, where any deletion removing the heterologous sequence should be viable. Third, in the case of the bicistronic genome, translation of the HCV polyprotein is under the control of the EMCV IRES, which may be undesirable in certain cases because any interference with the activity of this IRES impairs RNA replication and thereby mimics an antiviral effect.
One obvious application of the monocistronic GFP or RFP genomes is in the study of the dynamics of viral replication in living cells. Although subgenomic replicons carrying in-frame GFP insertions in analogous positions of NS5A have been described recently (2, 38), the advantage of this novel system is the possibility to characterize the dynamics of viral replication, and eventually the biogenesis of the HCV replication complex, upon (synchronized) infection. Moreover, upon coinfection of cells with viruses directing the expression of different fluorochrome reporter-NS5A fusion proteins, it is possible to study whether replication complexes derived from individual genomes remain separated from each other or intermingle. In fact, preliminary data obtained with this novel system suggest that mixed replication complexes do form, at least to some extent, but further studies are required to fully understand their biogenesis.
Taking advantage of the novel reporter virus system, we showed that cells previously infected with HCV are largely resistant to secondary HCV infection but not to infection with a heterologous virus, as exemplified for DEN-2. This superinfection exclusion is not due to a block at the level of virus entry, at least up to 120 h post-primary infection, which is different from the superinfection exclusion observed for several other viruses. For instance, in the case of HIV, the immature envelope glycoprotein gp160 forms a complex with CD4 (12, 23). Presumably due to poor intracellular transport of gp160, this complex is retained in the endoplasmic reticulum and contributes to down regulation of the primary CD4 receptor on the surfaces of infected T cells. In addition, the viral factors Vpu and Nef contribute substantially to CD4 receptor down regulation, although Nef mediates superinfection exclusion primarily by some other CD4-independent manner (see reference 51 and references cited therein). In the case of the pestivirus bovine viral diarrhea virus, superinfection exclusion appears to be mediated at two levels, namely, virus entry and RNA replication (32). While the entry block requires expression of the envelope glycoprotein E2, inhibition of replication of the incoming RNA is independent from the structural proteins. The underlying mechanisms are not yet understood.
While we did not observe a block of virus entry for up to 5 days after primary HCV infection, we cannot exclude the possibility that an entry block develops at later time points. We note that Zhong and coworkers found that during prolonged passage of cells infected with a cell culture-adapted JFH-1 variant, cells become largely resistant to HCV infection (54). This resistance is due to selection for cells that have small amounts of CD81 on their surfaces or that support HCV RNA replication with only a low efficiency. Since JFH-1 exerts a cytopathic effect (53), prolonged culture of infected cells most likely results in selection for those cells that are largely resistant to JFH-1 infection, either by down regulation of important entry molecules or by providing a hostile environment for RNA replication. These cells have a growth advantage over infected cells and therefore become predominant in the culture. In contrast, in our study, we characterized cells only early after primary infection and observed no such negative effects at the level of virus entry. In accordance with our FACS and infection analyses (Fig. 5), we therefore conclude that JFH-1 does not induce a block at the level of virus entry, or does so only to a minor extent, but rather forces selection of poorly infectible cells due to its cytopathogenicity.
By using supertransfection experiments with cells carrying stably replicating subgenomic HCV replicons, we found that translation and, presumably, replication of the transfected (secondary) RNA are impaired. This result is in full agreement with previous observations that the replication efficiency of an HCV RNA decreases in the presence of increasing amounts of a viral RNA cotransfected into the same cell (34). Moreover, upon transfection of increasing amounts of replication-competent viral RNA, the replication efficiency per transfected molecule decreases (34). It is therefore assumed that one or more cellular factors required for efficient HCV RNA replication are limiting in a cell. One would therefore expect that cells can support replication of HCV RNA only to a certain level and that the extent of superinfection exclusion directly correlates with the replication fitness of the primary infecting RNA. In fact, it was found that replication fitness of one replicon reduces the capacity of a second replicon to stably replicate in the same culture (16), arguing for a limiting host cell factor(s) and condition(s) that are required for efficient HCV RNA replication. Although similar findings have been obtained with bovine viral diarrhea virus (32), arguing for a common mechanism of superinfection exclusion at the level of RNA replication, alternative explanations are possible. For instance, large amounts of HCV protein(s) may exert a direct negative effect on replication of the incoming RNA. Such a scenario is assumed for Borna disease virus, where resistance to homologous secondary infection has been ascribed to unbalanced levels of viral nucleocapsid components (18). It is hypothesized that the incorrect stoichiometry of nucleocapsid components leads to an inhibition of the polymerase activity of incoming viruses. Alternatively, large amounts of HCV protein(s) may alter correct trafficking of the incoming superinfecting virus, as suggested in the case of the L surface antigen of the duck hepatitis B virus (50). In the case of Sindbis virus, expression of the nonstructural proteins is sufficient to block secondary infection at the level of RNA replication without affecting RNA translation (1, 22). It is assumed that large amounts of the viral protease accumulated in previously infected cells lead to rapid proteolytic cleavage of the replicase expressed from the incoming RNA, resulting in an inhibition of the negative-strand copy from the incoming RNA (27).
Thus far, the JFH-1 isolate is the only one that replicates to very high levels, whereas all other functional HCV clones described thus far require cell culture-adaptive mutations and even then replicate less efficiently (3). Given the likely correlation between superinfection exclusion and the replication fitness of the primary infecting genome, it is not clear how well the observations made with JFH-1 can be extrapolated to other HCV isolates. Even though interference at the level of RNA replication has also been described for replicons derived from the genotype 1b isolate Con1, these RNAs always carried replication-enhancing cell culture-adaptive mutations (16, 34). Thus, the questions of whether superinfection exclusion also occurs with isolates that are less fit in replication and how these isolates codevelop in long-term passages require the establishment of further functional HCV clones that replicate in the absence of adaptive mutations and that, ideally, support infectious virus production, too. For the same reasons, it is unclear to what extent superinfection exclusion occurs in vivo. Nevertheless, one obvious consequence would be that superinfection exclusion reduces the likelihood of recombination between two viral genomes coreplicating in the same cell. In fact, RNA recombination has been observed only sporadically, arguing that it is a rare event (11, 25). While on one hand superinfection exclusion reduces the likelihood of interference between two viral genomes in the same cell, on the other hand it reduces genetic plasticity by lowering the chance for the formation of novel virus recombinants. It remains to be determined how the complex interplay between replication fitness, superinfection exclusion, and RNA recombination is regulated.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (BA1505/2-1 to R.B. and N.A.), the Sonderforschungsbereich 638 (Teilprojekt A5 to R.B.), and the Forschungsschwerpunkt of the state Baden-Württemberg (Teilprojekt 1 to R.B. and Teilprojekt 4 to T.P.) and by an Emmy Noether scholarship from the Deutsche Forschungsgemeinschaft to T.P. (PI734/1-1).
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FOOTNOTES |
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Published ahead of print on 14 February 2007.
T.S. and N.A. contributed equally to this work.
Present address: Centre for Virology, Department of Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF, United Kingdom.
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