Viral and cellular determinants of hepatitis C virus RNA replication in cell culture - PubMed (original) (raw)
Viral and cellular determinants of hepatitis C virus RNA replication in cell culture
Volker Lohmann et al. J Virol. 2003 Mar.
Abstract
Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.
Figures
FIG. 1.
Conserved mutations in replicons isolated from 26 independently selected replicon cell clones. A schematic drawing of the HCV NS proteins as present in the subgenomic replicons is shown. Regions encoding known enzymatic functions are shaded. Mutations conserved in the replicon population of single cell clones are given above. Numbers refer to the amino acid position in the complete polyprotein of the HCV Con-1 isolate. The frequency with which a given mutation was found in these cell clones is indicated in parentheses. Mutations identified as the only conserved alteration in at least one cell clone are underlined. A summary of these data is given in Table 1. Ins, insertion; del, deletion; prot., proteinase.
FIG. 2.
Transient replication of replicons with different IRES elements at the 5′ end. (A) Structure of the replicons rep HI-luc and rep PI-luc used for transient replication assays. 5′, HCV 5′ NTR; 3′, HCV 3′ NTR; PI, PV IRES; luc, firefly luciferase; EI, EMCV IRES. (B) Transient replication of the replicons carrying the luciferase reporter gene under translational control of the HCV IRES or the PV IRES. Huh-7 cells were transfected with 5 μg of rep PI-luc/5.1, rep PI-luc/GND, rep HI-luc/5.1, or rep HI-luc/GND, and luciferase activity was determined in cell lysates that were prepared at given time points posttransfection. Data were normalized for transfection efficiency among constructs with identical IRES elements as determined by measurement of the luciferase activity 4 h after transfection. Electroporations and luciferase assays were performed in duplicates. Note the logarithmic scale of the ordinate. (C) Analysis of transient replication of different luciferase replicons by Northern hybridization. Huh-7 cells were transfected with 5 μg of luciferase replicons 5.1 that carry two cell culture-adaptive mutations in NS3 and one in NS5A (28) or with the ET replicons carrying the same NS3 mutations and one in NS4B instead of NS5A (Table 2). Cells were harvested at different time points postelectroporation (pE) as indicated above each lane. Then, 10 μg of total RNA was analyzed by Northern hybridization with a 32P-labeled negative-sense riboprobe spanning the NS3 to NS5B region. The positions of replicon RNAs with PV IRES (rep PI) or HCV IRES (rep HI) and β-actin mRNA are given on the right. (D) Quantification of the Northern hybridization shown in panel C by phosphorimaging. Values were corrected for different RNA amounts by using the β-actin signal and normalized for the PSL obtained at 4 h after transfection.
FIG. 3.
Effect of single amino acid substitutions in the HCV coding sequence on transient replication. Values represent the ratio of RLU measured at 48 h and at 4 h after electroporation. The 4-h value was set at 100%. Data are means and the standard deviations of at least three independent experiments. For all experiments, Huh-7 cells with passage numbers of >90 were used.
FIG. 4.
Cooperativity and incompatibility of adaptive mutations. Huh-7 cells were transfected with replicons containing the mutations specified in the left of each panel and harvested 4 and 48 h later. Luciferase activity is expressed as the percent RLU determined in lysates of cells at 48 h compared to those at 4 h after transfection. (A) NS3 mutations cooperatively enhance transient replication of replicons with NS4B, NS5A, and NS5B mutations. Pairs of replicons containing highly adaptive mutations without (gray bars) or with (black bars) two adaptive NS3 mutations are separated by dotted lines. The replication efficiency of the replicon carrying only the two NS3 mutations is given at the top. (B) Incompatibility of mutations in NS4B, NS5A, and NS5B. Replication efficiencies of replicons containing single highly adaptive mutations (gray bars) or combinations of these mutations (black bars) are compared. Replication levels of a wild-type replicon (wt) and a replication-deficient mutant (GND) are shown at the bottom of the panel (white bars). Values in both panels are means and standard deviations of at least three independent experiments. For all experiments, Huh-7 cells with passage numbers of >90 were used.
FIG. 5.
Influence of Huh-7 passage number on transient HCV replication. (A) Replicon PI-luc/ET (1 μg) was transfected into different passages of Huh-7 cells that were harvested after 4, 24, 48, and 72 h. Luciferase activity measured in the 4-h lysate was set as 100% for every passage tested. Data are from a representative experiment. Note the logarithmic scale of the ordinate. (B) Replicons carrying different mutations specified at the bottom were transfected into different passages of Huh-7 cells or cell clone 9-13 that was cured from the replicon by treatment with IFN-α (9-13cured). Luciferase activity is expressed as the percent RLU determined at 48 h compared to that determined at 4 h after transfection. Note the logarithmic scale of the ordinate.
FIG. 6.
Comparable translation from the EMCV and the PV IRES in different Huh-7 passages. Replication-deficient RNAs containing a luciferase gene under the translational control of an EMVC (A) or a PV IRES (B) as indicated at the top of each panel were electroporated into different passages of Huh-7 cells. In vitro transcripts coding for β-galactosidase were cotransfected to normalize for transfection efficiency. Cells were harvested 2 h after electroporation, and the luciferase and β-galactosidase activities were determined. The given values are corrected for transfection efficiency. Passage numbers of the Huh-7 cells used for transfection are indicated under each column.
FIG. 7.
Comparable stabilities of replicon RNAs in different Huh-7 passages. Cells specified in the top were transfected with 15 μg of the replication-defective replicon PI-luc/GND and harvested at different time points postelectroporation (pE) as indicated above each lane. (A) Total RNA was analyzed by Northern hybridization in parallel to known amounts of in vitro transcripts (control) and total RNA from naive Huh-7 cells (−). The positions of replicon RNA (HCV) and β-actin mRNA are given on the right. (B) Quantification of the Northern hybridization shown in panel A by phosphorimaging. Values were corrected for differences in loading by using the β-actin signal and are expressed as the percent PSL at a given time point relative to that observed 2 h after transfection.
FIG. 8.
Inverse correlation between the amount of transfected replicon RNA and the replication efficiency. Increasing amounts of rep luc/ET RNA were used for electroporation of Huh-7 cells. The amount of total transfected RNA in every sample was adjusted to 20 μg by the addition of yeast tRNA. Cells were harvested 4 and 72 h after electroporation and assayed for luciferase activity. The replication efficiency is expressed as the percent RLU determined at 72 h compared to that determined at 4 h after transfection. Note the logarithmic scale of the abscissa.
FIG. 9.
Coexpression of the HCV NS proteins, not the cotransfected RNA itself, affects RNA replication. (A) Schematic structure of RNAs used for cotransfection experiments with rep PI-luc/ET. Adaptive mutations are indicated by asterisks. “ΔGDD” refers to the position of the 10-amino-acid deletion spanning the GDD motif in NS5B, and “stop” refers to an engineered stop codon that inhibits translation ∼70 amino acids downstream of the NS3 inititation codon. For further details, see the legend to Fig. 2A. (B) Increasing amounts of rep PI-luc/ET-RNA (0.1, 0.4, 1.25, 5, and 20 μg) were cotransfected with different competitors specified at the top. In each case the total amount of transfected RNA was adjusted to 20 μg by the addition of decreasing amounts of competitor RNA (19.9, 19.6, 18.75, 15, and 0 μg). Huh-7 cells were electroporated with this mixture, seeded into aliquots, and harvested at 4 and 24 h after transfection. Replication efficiency is expressed as the percent RLU determined 24 h compared to 4 h after transfection. Note the logarithmic scale of the abscissa.
References
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