Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

Abstract

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (-) RNA synthesis have been identified in the 3' non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (-) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3'-end of the (-) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.

Figure 1.

Some mutations in the SL-E1 stem–loop decreased in vitro RNA synthesis by NS5B. (A) Secondary structure of SL-E1 stem–loop. Arrow heads indicate the position of the nucleotide changes. Sequence homologous to SLII stem–loop is filled in grey. (B) An RdRp assay was performed with the purified NS5B1b (200 nM) and mutant RNAs as templates as described in ‘Materials and Methods’ section. The amount of RNA synthesized was determined after TCA precipitation and counting in a Wallac scintillation counter. The results were expressed as the percentage of the value obtained with the WT RNA (mean ± SD, n = 3 independent experiments).

Figure 2.

Secondary structure models of WT and 2A, 4A and 2AU mutant 341 nt RNAs. WT RNA: The first 222 nt (numbered from 3′-end) of the 341 nt WT fold in five stem–loops identical to those proposed by Schuster et al. (21) and Smith et al. (22). The same notation as in (21) was used. The 223–317-nt sequence fold in a structure similar to the IIIcdef’ structure presented by Smith et al. (22). Lead reactivity is represented by triangles, RNase T1 reactivity is represented by circles and DEPC reactivity is represented by squares. Empty or filled symbols are for weak or strong cleavage, respectively. 2A, 4A and 2AU RNAs: a close-up section of the model secondary structures of nt 156 to nt 317 are shown. Reactivity is indicated by the same symbols. The positions of A and U residues introduced by mutations of G or C residues are indicated by arrows.

Figure 3.

Some mutations in the SL-E1 stem–loop decreased initiation of RNA synthesis. For the single-round replication assay, HCV RdRp and RNA were pre-incubated for 30 min at 25°C in the reaction mixture without ATP and UTP. Heparin (MW 4000–6000 Da, 200 µg/ml) was then added followed by ATP and [³²P]UTP. The reaction mixture was further incubated at 25°C for 0, 5, 10, 20 and 60 min. The ³²P RNA products were (A): quantified after TCA precipitation and counted in a Wallac counter (filled circles, WT RNA; open circles, 2AU RNA; filled triangles, 2A RNA; empty triangles, 4A RNA) or (B): analyzed on denaturing polyacrylamide gels. For the gel-base initiation assay, an RdRp assay was performed with 0.5 mM GTP and 10 µM CTP with 4 µCi [α-³²P] CTP (3000 Ci × mmol^–1) as only added NTP. The reaction was run at 25°C for 10, 30 and 60 min. Initiation products were run in a 20% acrylamide gel in TBE buffer, submitted to electronic autoradiography using a Pharos apparatus (Biorad) and quantified using the Quantity one software (Biorad). (C) Quantification of initiation products synthesized with WT and mutant RNAs after 1 h incubation. Data correspond to the mean of three independent experiments ± SD. (D) Analysis of initiation products on polyacrylamide gel electrophoresis. M: pppGpC molecular weight marker.

Figure 4.

Effect of mutations on HCV IRES activity. (A) Secondary structure of HCV 5′UTR according to Honda et al. (45). Nucleotide changes corresponding to 2A, 4A and 2AU mutants are indicated by arrows. The sequence complementary to the apical loop of SLII of the 3′UTR is underlined in grey. (B) Schematic representation of bicistronic RNA produced from pIRF vector. (C) Bicistronic RNAs transcribed from pIRF vector containing WT or mutated HCV 5′UTR were transfected into Huh7-QR cells. The activity of each IRES was determined by calculating the ratio of RLuc to FLuc activities in cell extracts. Values are expressed as the percentage of the relative activity of the WT IRES. Each bar represents the mean value obtained in three independent experiments ± SD.

Figure 5.

Some mutations in the SL-E1 stem–loop decreased RNA synthesis by RC. (A) Schematic representation of polycistronic RNA produced from pGEM-T/5UTR-H2AE-3UTR. FMDV prot is for FMDV 2A protease. (B) Polycistronic RNAs transcribed from pGEM-T/5UTR_H77-H2AE-3UTR_H77 WT or 2A or 5UTR-EGFP-3UTR were transfected into Huh7/Rep5.1 cells or Huh7-QR. After a 3-week selection, cell colonies were numbered under microscopy examination. Huh7/Rep5.1 cells transfected with 5UTR_H77-H2AE-3UTR_H77 WT RNA, (1), 5UTR_H77-H2AE-3UTR_H77 2A RNA (2), 5UTR-EGFP-3UTR RNA (3) and Huh7-QR cells transfected with 5UTR_H77-H2AE-3UTR_H77 WT RNA (4). (C) The number of hygromycin-resistant colonies was determined after transfection of WT and mutated 5UTR_H77-H2AE-3UTR_H77 minigenomes in Huh7/Rep5.1 cells. Values were expressed as the percentage of the colony number obtained with the minigenome containing the WT 5′UTR. Each bar represents the mean value obtained from at least three independent experiments ± SD. (D) Northern blot analysis. RNAs (10 µg) of transfected Huh7/Rep5.1 cells were fractionated on 1% denaturing agarose gel and transferred to nylon membrane. Membrane-bound RNAs were hybridized with EGFP or GAPDH probes. Mock transfected cells (lane 1), cells transfected with WT minigenome (lane 2), cells transfected with 2A minigenome (lane 3), cells transfected with 2GA minigenome (lane 4), cells transfected with SLE1AL minigenome (lane 5).