Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication

Abstract

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.

FIG. 1

A schematic presentation of the basic replicon construct used in this study is at the top. The 5′ NTR indicated with a thick line is positioned upstream of either the gene encoding the neomycin phosphotransferase (neo) or the luciferase (luc) of the firefly Photinus pyralis. The EMCV IRES (EI) directs translation of the NS3 to NS5B region that is flanked at the 3′ end by the 3′ NTR (thick line). ∗, positions of cell culture-adaptive mutations. A schematic presentation of the secondary structure of the HCV 5′ NTR according to a previous study (18) is drawn below. Framed numbers refer to stem-loops 1 to 4. The borders of the HCV IRES are indicated with dotted lines. Numbers and arrows refer to the 3′ boundaries of the HCV sequences of 5′ NTR chimeras. ●, initiator AUG codon of the HCV polyprotein with the A residue at nucleotide position 342 located in stem-loop 4.

FIG. 2

Effect of sequences upstream of HCV IRES on translation of replication-competent bicistronic replicon RNAs. (A) Schematic presentation of the 5′ NTRs of the various replicons. (B) In vitro translation of replicon RNAs in extracts of rabbit reticulocytes (left), Huh-7 (middle), and HeLa (right) cells. Lysates were programmed with either an RNA encoding the NPT under control of the poliovirus IRES (lane neo) or the replicon RNAs given above the lanes. An RNA encoding only the NS3 under control of the EMCV-IRES was used to generate authentic NS3 as a size marker. The background was determined with translation reaction mixtures without in vitro transcript (no RNA). Note that the replicon RNAs direct the expression of a C-NPT fusion protein that carries 16 amino acid residues of the core protein at its amino terminus. Numbers to the left refer to the sizes of molecular mass standards in kilodaltons; proteins are specified to the right. Numbers below the lanes refer to the translation levels of the C-NPT fusion protein after normalization for the amount of NS3. Translation efficiency obtained with the replicon carrying the full-length HCV IRES (389) was set at 1. Representative results are shown. (C) Translation efficiencies after transfection of Huh-7. Cells were transfected with either of the replicon constructs specified in the bottom together with an RNA encoding β-Gal under control of the EMCV IRES. Four hours after transfection, cells were lysed and reporter activities were determined as described in Materials and Methods. Luciferase activity obtained with the parental replicon 389 was set at 100%. Mean values of three independent experiments, and the error ranges are given.

FIG. 3

Sequences upstream of stem-loop 2 are essential for RNA replication. (A) Result of transient-replication assay with replicons carrying firefly luciferase gene. Huh-7 cells were transfected with RNAs (given at the bottom), and luciferase activities were measured 24, 48, and 72 h later. Values are normalized for transfection efficiency as determined from the luciferase activities measured 4 h after transfection (set at 100%). The experiment is a representative example of three repetitions. Values are means of quadruplicates and standard deviations. The result obtained with an RNA with a full-length HCV 5′ NTR and an inactive NS5B RdRp is shown on the right (389/GND). (B) Selectable replicons carrying deletions in the first 40 nucleotides of the HCV 5′ NTR are unable to confer G418 resistance. Huh-7 cells were transfected with either 1 ng of the selectable replicon carrying the first 389 nucleotides of the HCV genome (389) or with each 100 ng of the deletion mutants. Twenty-four hours later, cells were subjected to G418 selection, and after about 3 weeks, colonies were fixed and stained. Transfection of 100 ng of the replicon with the unaltered 5′ NTR and an inactive RdRp in which 10 amino acid residues spanning the active site were deleted served as a negative control (389/ΔGDD).

FIG. 4

(A) Schematic presentation of the replicon constructs with HCV-PV chimeric 5′ NTRs. HCV sequences in the 5′ NTR are drawn as a thick dotted line, the 63-nucleotide-long spacer (sp) element is a black box, and the PV IRES (PVI) is an open arrow. (B) Detailed presentation of the various chimeras. The parental construct with the full-length HCV 5′ NTR is framed. The two constructs in which the apical stems of stem-loop 2 were removed were derived from construct 125-sp-PVI (arrows).

FIG. 5

Translation studies of replicons with HCV-PV chimeric 5′ NTRs in vitro and in transfected cells. (A) Equal amounts of in vitro transcripts in which various portions of the HCV 5′ NTR were fused via the 63-nucleotide spacer to the IRES of PV were used to program HeLa cell extracts as described in Materials and Methods. Translation reactions were terminated by the addition of protein sample buffer, and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins are specified on the right, the positions of molecular mass standards (kilodaltons) are indicated on the left. The result obtained after in vitro translation of the parental replicon with the authentic HCV 5′ NTR is shown in lane 3 (rep5.1). Note that this RNA encodes a C-NPT fusion protein whereas the neo gene is directly fused to the PV IRES in the case of the replicons with the chimeric 5′ NTRs. For comparison, an RNA containing the neo gene under the control of only the PV IRES was translated in parallel (lane 2). Lane 1, background bands obtained with the cell extracts without exogenous RNA. (B) Translation efficiencies of the HCV-sp-PV chimeric RNAs in cells. Huh-7 cells were transfected with luciferase replicons (specified at the bottom) together with an RNA encoding β-Gal. Four hours after transfection, cells were lysed and luciferase activities were determined. Values are corrected for transfection efficiencies as determined by measuring β-Gal activities in the same cell lysates. Mean values of four independent experiments and the error ranges are given. Values obtained with the parental replicon, 341-sp/wt, were set 100%. The results obtained with the replication defective RNAs that contain or lack the spacer between the HCV 5′ NTR and the PV IRES are shown in the right (341-sp/GND and 341/GND, respectively). Only a representative selection of the data obtained with the chimeric RNAs is shown.

FIG. 6

The complete HCV 5′ NTR is required for efficient RNA replication. (A) Replicons with chimeric HCV-sp-PV 5′ NTRs directing the expression of the luciferase gene were transfected into Huh-7 cells. After 4 (not shown), 24, 48, and 72 h, cells were lysed and luciferase activities were determined. The 4-h value that was set at 100% was used to correct for different transfection efficiencies. A representative result of three independent experiments is shown. Values represent the mean of quadruplicates and the standard deviation. (B) Huh-7 cells were transfected with selectable RNAs in which the neo gene was under the control of the different chimeric 5′ NTRs. About 3 weeks after transfection and G418 selection, cells were fixed and stained with Coomassie brilliant blue. The CFU/microgram of in vitro transcript as determined by transfection of serial dilutions of RNA is given below each culture dish. ΔGDD, inactive replicon with a 10-amino-acid residue deletion spanning the active site of the NS5B RdRp (28).