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. 2003 Feb;77(3):2029-37.
doi: 10.1128/jvi.77.3.2029-2037.2003.

Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells

Richard W Hardy  1 , Joseph Marcotrigiano, Keril J Blight, John E Majors, Charles M Rice
Affiliations

Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells

Richard W Hardy et al. J Virol. 2003 Feb.

Abstract

A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40 degrees C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn2+ and Mg2+ cations could be used in the reaction; however, concentrations of Mn2+ greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.

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Figures

FIG. 1.
FIG. 1.
Replicon structure and fractionation scheme. (A) Schematic representation of the HCV subgenomic replicon RNA. The replicon contains the 5′- and 3′-UTRs of HCV and two cistrons encoding the first 12 amino acids of core fused to neomycin phosphotransferase (Neo) and NS3-NS5B. The HCV IRES located within the 5′-UTR and the EMCV IRES initiate translation of Neo and NS3-NS5B, respectively. (B) Procedure for isolation of membrane fractions from replicon-containing Huh7 cells. Cellular membranes were isolated by lysing cells in a hypotonic buffer, followed by two centrifugation steps. The cell lysate was centrifuged at 900 × g to pellet nuclei and unlysed cells (P9 fraction). The supernatant (S9) was further fractionated by centrifugation at 15,000 × g. The resulting supernatant (S15) and pellet (P15) were enriched for cytoplasm and cellular membranes, respectively.
FIG. 2.
FIG. 2.
Locations of HCV proteins and replicase activity in the P15 fractions. (A) During the isolation, samples were prepared from each step and subjected to Western blot analysis with antisera directed against NS3, NS5A, and NS5B. NS5A appears as two bands (p56 and p58) for the basal and hyperphosphorylated forms. The majority of HCV proteins appear to be found in the P9 and P15 fractions. For comparison, equivalent amounts were loaded onto each lane. (B) Membrane fractions (P15) from replicon-containing cells (+) and Huh7 cells (−) were assayed for replicase activity. P15s were incubated with [32P]CTP in the absence (−) or presence (+) of a full complement of NTPs (ATP, GTP, and UTP). Denatured products were separated on a 1% agarose gel and visualized by autoradiography. Radiolabeled replicon RNA marker (lane M) was generated by in vitro transcription by T7 RNA polymerase of a linearized DNA template.
FIG. 3.
FIG. 3.
Temperature range and time course of cell-free HCV RNA synthesis. Production of HCV RNA from P15 fractions under standard conditions (Materials and Methods) was assessed over a range of temperature (A) and time (B). RNA products were extracted, precipitated, and analyzed by denaturing agarose gel electrophoresis. (A) Reactions were incubated at 25, 30, 34, 37, and 40°C for 1 h and then terminated by the addition of SDS and proteinase K. P15 fractions from Huh7 cells lacking the replicon (−) were assayed at 34°C as a control for background RNA synthesis. (B) P15 fractions from replicon-containing cells were incubated at 34°C for 0, 7, 15, 30, 45, 60, 90, and 120 min under standard conditions. At each time point the reactions were stopped immediately by freezing in a dry ice bath prior to RNA extraction.
FIG. 4.
FIG. 4.
Divalent cation requirements for HCV RNA synthesis. In vitro RNA replication reactions were carried out in the presence of various concentrations (0.1 to 10 mM as indicated) of Mn2+ (A) or Mg2+ (B) at 34°C for 1 h. RNA products were extracted and analyzed by denaturing agarose gel electrophoresis (Materials and Methods), and the position of the full-length replicon RNA is indicated. (A) An asterisk denotes the position of an RNA that was smaller than the replicon that was formed with the addition of 0.25 to 1 mM Mn2+ to the reaction mixture. (B) Synthesis of RNA over the range of Mg2+ concentrations is compared to the same reaction done in the presence of the optimal Mn2+ concentration (1 mM).
FIG. 5.
FIG. 5.
Polarity of HCV RNA products. RNase H digestion was employed as a means of determining the polarity of the RNA replication products. DNA oligonucleotides were designed to specifically hybridize to either plus-sense (+) or minus-sense (−) replicon RNA. (A) Diagram illustrating the locations of annealing oligonucleotide and predicted products after RNase H digestion. Oligonucleotide A should anneal to nt 3102 to 3121 of plus-strand replicon RNA; oligonucleotide B corresponds to nt 2081 to 2100 of the plus-sense replicon RNA and is predicted to anneal to minus-strand RNA. After hybridization of the DNA oligonucleotides, followed by RNase H digestion, the polarity of the RNA is determined by the production of products 1 and 2 for the plus (+) sense and products 3 and 4 for the minus (−) sense. (B) Products of RNase H digestion. RNase H digestions were performed on T7 transcribed RNA (left panel) and the RNA products of the cell-free reaction (right panel). Hybridizing DNA oligonucleotides used in the digestion are denoted by A and B. Two RNA markers of 6274 and 2024 nt were synthesized by in vitro transcription of a linearized DNA template. Oligo, oligonucleotide.
FIG. 6.
FIG. 6.
Evidence for de novo initiation of RNA replication. (A) Heparin sensitivity of the in vitro replication reaction. Standard reactions were performed on P15s from Huh7 (− replicon) and clone A (+ replicon) cells in the presence (+) or absence (−) of 4 ng of heparin/μl. (B) Standard P15 reactions (represented by “−”) were performed with 1 mM ATP, GTP, or UTP; 40 μM CTP; and 1 mCi of [α-32P]CTP per ml. Nucleotide requirements for replicon RNA synthesis were tested by limiting the concentration (2 μM) of either GTP, ATP, or UTP (denoted as G, A, and U in the figure). Nucleotide incorporation was determined on the basis of denaturing agarose gel electrophoresis (top panel) or average trichloroacetic acid-precipitable counts from three independent experiments (bottom panel).
FIG. 7.
FIG. 7.
Inhibition of HCV RNA synthesis by putative antiviral compounds. Various concentrations of NS3 and NS5B inhibitors were added to the membrane fractions and incubated at 25°C for 5 min prior to the addition of the other in vitro replication reaction components. Reactions and RNA product analysis were done by using standard conditions (Materials and Methods). (A) Chemical structures of the NS3 and NS5B inhibitory compounds. (B) P15 RNA synthesis reaction done in the presence of helicase inhibitor (left panel) or polymerase inhibitor (right panel). (C) The inhibitor titration experiments were performed in triplicate, quantitated by using a phosphorimager, and demonstrated graphically.
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