Cell-free replication of the hepatitis C virus subgenomic replicon

Abstract

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.

FIG. 1.

Genetic organization of the HCV subgenomic replicon. The bicistronic RNA molecule contains the HCV IRES and a few core-coding sequences, which are followed by neomycin phosphotransferase gene (Neo^r). These sequences are followed by the encephalomyocarditis virus IRES placed in front of HCV sequences encoding nonstructural protein NS3 to NS5B, terminating at the HCV 3′ NCR as described by Lohmann et al. (23).

FIG. 2.

(A) Cell-free HCV replication assay. FCA4 (lanes 2 to 5) or Huh-7 (lanes 6 to 9) lysates were incubated with [³²P]CTP for 10, 30, 60, or 90 min as indicated. The RNAs from these lysates were purified and fractionated by agarose gel electrophoresis in Tris-acetate-EDTA buffer. The gel was stained with ethidium bromide to visualize the RNA bands. The band intensities of rRNAs in each lane were found to be similar. Lane 1, in vitro-transcribed replicon that was labeled with the [α³²P]CTP and used as an RNA marker. The labeled products are indicated as replicative forms (RI/RF). ssRNA, position of replicon. The gel was dried and exposed to X-ray film for 14 h. (B) Determination of the molecular weight of the HCV replication products synthesized in the FCA4 lysates. The replication assay was carried out with FCA4 lysates as described above, and the migration of replication products was compared with that of the in vitro-labeled BM4-5 RNA marker. The samples were denatured and electrophoresed on a methylmercury agarose gel, and the gel was dried and autoradiographed.

FIG. 3.

RNase A sensitivity of the replication products. (A) RNA replication reactions, performed using FCA4 lysates. RNA products from the reactions were purified and resuspended in 20 μl of buffer containing 150 mM NaCl. The samples were treated with RNase A (DNase and protease free; 140 ng/ml) for 0, 1, 2.5, or 8 min at room temperature. The digestion was terminated by phenol extraction as described in Materials and Methods. The final products were resuspended in water and fractionated by 1% agarose gel electrophoresis to visualize RFs and RIs. The gel was dried and subjected to autoradiography. (B) Determination of the RNase A sensitivity of the replication products was carried out as described for panel A except that the RNase A concentration was raised to 1 μg/ml in this assay. (C) Determination of the stability of the HCV replicon in the cell-free replication system. In vitro-transcribed BM4-5 RNA (3 μg) was incubated in FCA4 replication lysates (lanes 2 to 7) for 0, 1, 2.5, 5, 10, and 15 min. The RNA was extracted from each of the samples and analyzed on a native (1%) agarose gel.

FIG. 4.

Effects of actinomycin D and micrococcal nuclease on HCV replication. (A) HCV replication assay of lysates from transiently transfected Huh-7 cells with HCV subgenomic replicon RNA (BM4-5) and FCA4 cell lysates treated with micrococcal nuclease. Agarose gel electrophoresis of the products of a replication assay carried out as described in Materials and Methods with lysates prepared from Huh-7 cells (lane 1), Huh-7 cell lysates transiently transfected with BM4-5 RNA subgenome (lane 2), FCA4 cell lysates (lane 3), FCA4 lysates treated with Ca²⁺ and subsequently with EGTA in a mock reaction (lane 4), and FCA4 cell lysates treated with micrococcal nuclease and Ca²⁺ and subsequently with EGTA (lane 5) is shown. (B) Replication of HCV RNA is resistant to actinomycin D. Agarose gel electrophoresis of the products of a replication assay with Huh-7 cell lysates (lane 1), FCA4 cell lysates (lane 2), and FCA4 cell lysates in the presence of actinomycin D (Act. D) (lane 3) is shown. (C) Analysis of the products synthesized in the replication assay. The RNAs labeled in Huh-7 (lane 1) or FCA4 (lane 2) cell lysates were fractionated with 2 M LiCl as described in Materials and Methods. The insoluble and soluble fractions were analyzed by native agarose gel electrophoresis. Lanes 3 and 4, LiCl-soluble forms of the replication assay products; lanes 5 and 6, nucleic acid products that are insoluble in LiCl.

FIG. 5.

Northern blot hybridization analysis of replication products. The in vitro-transcribed unlabeled RNAs representing the HCV subgenomic replicon (lane 1), the N-terminal half of the NS5A-coding region with plus (lane 2) or minus (lane 3) polarity, a nonspecific HBV RNA (lane 4) with a length similar to that of NS5A, and Huh-7 cytoplasmic RNA (lane 5) were fractionated by formaldehyde-agarose gel electrophoresis. The integrity of RNAs was determined by ethidium bromide staining before transfer to the nylon membrane. The ³²P-labeled replication products synthesized in FCA4 lysates were eluted from a separate agarose gel and used to probe the blot. The blot was autoradiographed for 2 days.