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Comparative Study
. 2007 May;81(9):4591-603.
doi: 10.1128/JVI.02144-06. Epub 2007 Feb 14.

Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes

Torsten Schaller  1 , Nicole Appel, George Koutsoudakis, Stephanie Kallis, Volker Lohmann, Thomas Pietschmann, Ralf Bartenschlager
Affiliations
Comparative Study

"V体育2025版" Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes

Torsten Schaller et al. J Virol. 2007 May.

Erratum in

  • J Virol. 2007 Jul;81(13):7327

Abstract (V体育官网入口)

Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of an infectious cell culture system using the genotype 2a isolate JFH-1. Taking advantage of this system in the present study, we investigated whether HCV infection leads to superinfection exclusion, a state in which HCV-infected cells are resistant to secondary HCV infection. To discriminate between viral genomes, we inserted genes encoding fluorescent proteins in frame into the 3'-terminal NS5A coding region. These genomes replicated to wild-type levels and supported the production of infectious virus particles. Upon simultaneous infection of Huh-7 cells, co-replication of both viral genomes in the same cell was detected. However, when infections were performed sequentially, secondary infection was severely impaired. This superinfection exclusion was neither due to a reduction of cell surface expression of CD81 and scavenger receptor BI, two molecules implicated in HCV entry, nor due to a functional block at the level of virus entry. Instead, superinfection exclusion was mediated primarily by interference at the level of HCV RNA translation and, presumably, also replication. In summary, our results describe the construction and characterization of viable monocistronic HCV reporter genomes allowing detection of viral replication in infected living cells VSports手机版. By using these genomes, we found that HCV induces superinfection exclusion, which is primarily due to interference at a post-entry step. .

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Figures (VSports)

FIG. 1.
FIG. 1.
Genomic HCV constructs used in this study and their RNA replication capacities. (A) Schematic diagram of the NS5A structure according to reference . An amphipathic α-helix (AH) contributing to NS5A membrane attachment resides at the N terminus. The three domains are indicated by shaded boxes and are separated by trypsin-sensitive regions with presumably low structural complexity (low-complexity sequence [LCS]). The site of insertion of the GFP gene between codons 382 and 383 of the NS5A coding region is indicated (shaded bar), as are the flanking amino acid sequences. (B) Structures of HCV constructs used in this study. The chimeric genome Jc1 is composed of the indicated genome fragments of the two genotype 2a isolates J6CF and JFH-1 fused at a distinct position within NS2 (42). With the exception of JFH1ΔE/GFP, which contains only JFH-1 sequences, all other genomic constructs used in this study were derived from Jc1. The regions encoding fluorescent proteins (GFP, RFP, and Venus-GFP), either inserted into the NS5A coding sequence or expressed from a separate cistron (Venus-Jc1), are shown as striped boxes. The mutant JFH1ΔE/GFP comprises a large in-frame deletion encompassing most of the coding region of E1 and E2. EI, EMCV IRES. (C) RNA replication of HCV genomes in transfected Huh7-Lunet cells, as determined by Northern blot analysis (upper panel). Total RNA was prepared from transfected cells at the time point specified above each lane, and HCV RNA was detected by Northern hybridization, whereas the lower part of the blot was probed with a β-actin-specific RNA probe. The given amounts of HCV in vitro transcripts of a subgenomic replicon spiked into total RNA of naive Huh7-Lunet cells were used as a positive control (lanes 1 to 3 and 17 to 19), total RNA of naive cells served as a negative control (lane 4 and 20), and a replication-deficient JFH-1 genome (JFH1/GND) carrying a mutation in the active site of NS5B served as a negative control for RNA replication (lanes 29 to 32). HCV-specific signals were quantitated by phosphorimaging and, after normalization for RNA amounts loaded into the gel, HCV RNA copy numbers were calculated (lower panel). Levels and kinetics of RNA replication of the monocistronic RNAs were comparable to those of wild-type Jc1, whereas the bicistronic genome Venus-Jc1 replicated to much lower levels and with slower kinetics.
FIG. 2.
FIG. 2.
Virus release from transfected Huh7-Lunet cells, as determined by core-specific ELISA. (A) After transfection of in vitro transcripts of the HCV genomes specified at the top, cells were lysed at the time points given, and core protein amounts accumulated in the cells were determined (upper panel). Cell culture supernatants harvested from these cells were analyzed by core ELISA in parallel (lower panel). (B) Efficiency of core protein release from cells transfected with the monocistronic genome Jc1/GFP, the bicistronic genome Venus-Jc1, the parental wild type (Jc1), or the envelope gene deletion mutant (JFH1ΔE/GFP). The percentage of released core protein in relation to total core protein (the sum of intra- and extracellular core protein) was calculated for each time point. Core release from cells transfected with monocistronic genomes carrying the insertion in NS5A was strongly reduced. Note that core release obtained with the ΔE mutant was below the detection limit and therefore marked with an asterisk. Data from a representative experiment of three independent repetitions are shown.
FIG. 3.
FIG. 3.
Infectivity of virus particles generated with chimeric genomes that express GFP. (A) Titers of infectious virus contained in supernatants of Huh7-Lunet cells at 24 h and 48 h posttransfection. Infectivity titers were determined by TCID50 assay. Mean values and error ranges for duplicates are shown. (B) Detection of GFP by autofluorescence in cells 48 h after transfection with viral genomes (upper row) or 72 h after infection (lower row) with the viruses specified at the top. In the case of Jc1, NS5A was detected by immunofluorescence (IF). Note the endoplasmic reticulum-like staining in the case of Jc1 and the monocistronic Jc1/GFP genome, in contrast to the largely nuclear Venus-GFP staining in the case of the bicistronic genome Venus-Jc1. Also note the divergent infectivity titers of the virus chimeras, as evidenced by the number of cells displaying green fluorescence (autofluorescence or NS5A-specific staining; lower row). (C) Detection of intracellular GFP expression by FACS analysis of infected cells. Naive Huh7.5 cells were inoculated with culture supernatant harvested from transfected Huh7-Lunet cells at 24 h (upper row) or 48 h (lower row) posttransfection, and 72 h after infection of Huh7.5 cells, they were fixed and GFP expression was determined by FACS analysis. HCV constructs used for transfection of Huh7-Lunet cells are specified at the top. The number in the lower right corner of each diagram refers to the percentage of cells expressing GFP. Note that Jc1 does not express GFP and therefore determines the background of the FACS assay.
FIG. 4.
FIG. 4.
Evidence for homologous but not heterologous superinfection exclusion. (A) Schematic of the experimental procedure. Cells were either doubly infected with Jc1/GFP and Jc1/RFP (I) or first infected with Jc1/GFP and 24 h or 48 h later challenged with Jc1/RFP (II and III, respectively). The lower panel shows a quantification of cells infected with only Jc1/GFP (green portion of each column), only Jc1/RFP (red portion of each column), or both viruses (yellow portion of each column). Values written in the columns refer to the percentages of all infected cells displaying either red, green, or both fluorescent signals. The total number of infected cells was set to 100%. The proportion of the total cell population that was HCV infected is given by the height of the respective column. Values refer to one of three independent experiments. In each experiment, 250 cells were counted for each time point. (B) Representative views of infected cells examined by fluorescence microscopy. Cells were analyzed for GFP or RFP fluorescence; nuclei were stained with DAPI (blue). White arrows refer to cells infected with both viruses. Note that after 24 h, doubly infected cells were observed only sporadically. (C) Superinfection exclusion is also observed after primary infection with the Jc1 wild type. The schematic displayed in the upper panel is analogous to that in panel A, except that Jc1 wild-type virus, which produces much higher titers of infectious virus than the reporter viruses and spreads rapidly in cell culture, was used for primary infection. The quantification of cells infected with Jc1/GFP after simultaneous infection with Jc1 (I) or 24 h or 48 h after primary infection with Jc1 (II and III, respectively) is shown in the lower panel. Values were obtained after counting 800 cells for each time point. (D) Analysis of cells by immunofluorescence (in the case of Jc1, which does not contain a reporter gene) or by autofluorescence (in the case of Jc1/GFP). The top row shows cells infected only with Jc1/GFP (left panel) or only with Jc1 (middle panel). Mock-infected cells are shown in the right panel. Nuclei were stained with DAPI. The panels below show representative sections of images in which Jc1/GFP (left lane) or Jc1 (middle lane) was detected. White arrows indicate cells infected with both viruses. Note the decreasing number of Jc1/GFP-infected cells already 24 h after primary infection with Jc1 (II). (E) No block of DEN-2 by prior HCV infection. Huh7.5 cells were either infected with HCV (Jc1) or mock treated, and 24 h later, cells were either inoculated with DEN-2 or mock treated. Twenty-four hours after DEN-2 infection, cells were analyzed for HCV and DEN-2 antigens by NS5A (red) and NS5 (green) immunofluorescence, respectively. The upper panels (from left to right) show only DEN-2 infection, only HCV infection, HCV and DEN-2 coinfection, and mock-infected cells. Note that the number of DEN-2-infected cells between the first and the third panel is unchanged irrespective of the HCV infection. The lower panels represent DAPI staining of the nuclei (blue).
FIG. 5.
FIG. 5.
HCV-infected cells express normal levels of CD81 and SR-B1 on the cell surface and are fully permissive for HCVpp superinfection. (A) Huh7.5 cells were infected with Venus-Jc1 (lower row) or were left untreated (upper row) and then were used for cell surface staining of CD81 (middle column) or SR-BI (right column). Cells incubated with only the secondary antibody (anti-mouse-allophycocyanin; left column) were used to set the background. Venus-GFP expression in infected cells and cell surface staining were detected by FACS analysis. Note that the amounts of cell surface-expressed CD81 and SR-BI were unchanged in HCV-infected cells. (B) Naive Huh7.5 cells and cells previously infected with Jc1 (Jc1 inf) were inoculated with pseudoparticles carrying an HCV envelope from Con-1, JFH-1, or J6CF (HCVpp) or the VSV-G envelope (VSV-Gpp) or with lentiviral particles produced in the absence of a viral envelope glycoprotein (no env). Cells were harvested 72 h after secondary infection and analyzed by assessing luciferase activity expressed from the transducing lentiviral vector. Mean values for duplicate wells, with standard deviations, are shown. The panel on the right displays Jc1-infected and naive Huh7.5 target cells that were fixed and analyzed for NS5A expression by indirect immunofluorescence.
FIG. 6.
FIG. 6.
Superinfection exclusion is primarily due to interference at the level of RNA translation/replication. (A) Schematic diagrams of the bicistronic replicons used in this experiment. The insertions of the RFP and GFP genes into NS5A are indicated by colored boxes, the neo and firefly luciferase (Luc) genes are indicated by open boxes, and the EMCV IRES is indicated by a small dark gray box. (B) Naive Huh7-Lunet cells, a Huh7-Lunet cell pool carrying a stably replicating sg/neo/5A-RFP replicon, or cured sg/neo/5A-RFP replicon cells that no longer express detectable amounts of HCV proteins were transfected with the luciferase replicon, and luciferase activity was measured at the given time points posttransfection. The luciferase activity determined 4 h after transfection was set to 1. Error bars represent standard errors of the means for eight measurements from four independent wells in two independent experiments. RLU, relative light units. (C) Huh7-Lunet cells carrying the sg/neo/5A-RFP replicon, cured sg/neo/5A-RFP cells, and Huh7-Lunet cells were transfected with the sg/luc/5A-GFP replicon and analyzed by fluorescence microscopy at 48 h posttransfection. Results obtained with subgenomic replicon cells are shown in the upper row, with naive Huh7-Lunet cells shown in the bottom row and cured replicon cells shown in the middle row. Note that the number of GFP-expressing cells was much higher in the case of naive Huh7-Lunet cells and cured replicon cells than in neo replicon-containing cells (60% versus 5% in the experiment shown).
FIG. 7.
FIG. 7.
Evidence for interference at the level of RNA translation. (A) Structures of transfected HCV RNA sg/luc/JFH1/ΔGDD and the capped Renilla luciferase-encoding RNA. (B) A pool of Huh7-Lunet cells carrying autonomously replicating sg/neo/5A-RFP replicons (rep) or a cured pool of these cells (cured) were cotransfected with the replication-deficient subgenomic firefly luciferase replicon (Sg/luc) and the capped Renilla luciferase RNA (Rluc). Cells were harvested at the indicated time points, and firefly luciferase and Renilla luciferase activities were measured in cell lysates. Error bars represent standard errors of the means for eight measurements from four independent wells in two independent experiments.
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VSports注册入口 - References

    1. Adams, R. H., and D. T. Brown. 1985. BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus. J. Virol. 54:351-357. - PMC - PubMed
    1. Appel, N., T. Pietschmann, and R. Bartenschlager. 2005. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J. Virol. 79:3187-3194. - PMC - PubMed
    1. Bartenschlager, R. 2006. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture. Curr. Opin. Microbiol. 9:416-422. - VSports最新版本 - PubMed
    1. Bartenschlager, R., M. Frese, and T. Pietschmann. 2004. Novel insights into hepatitis C virus replication and persistence. Adv. Virus Res. 63:71-180. - PubMed
    1. Bartosch, B., J. Dubuisson, and F. L. Cosset. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197:633-642. - PMC - PubMed

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