The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site - PubMed (original) (raw)

The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site

T Ito et al. J Virol. 1998 Nov.

Abstract

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3'-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5'-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3'-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5'-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3' end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.

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Figures

FIG. 1

Functional analysis of the HCV X region by in vitro translation. (A) Schematic diagrams of pHCV-5CL and its related plasmids used in this study. pHCV-5CL contains T7 promoter (large open arrow), the 5′-UTR (single line), and core-encoding region (open box) of the HCV 1b strain fused to LUC genes (closed box) in the pGL vector (Promega). pHCV-5CL-X, -Xa, and -Xg contain, in addition, the X region and its mutants, respectively, at the 3′ end. The plasmids were linearized with the appropriate restriction enzymes and transcribed with T7 RNA polymerase to generate transcripts. (B) Computer-predicted secondary structures of the X region and its mutants in HCV-5CL-X, -Xa, and -Xg RNAs (17). SL2, stem-loop 2. (C) In vitro translation products of various RNAs separated by SDS-PAGE on 7.5% polyacrylamide gels. In vitro translation was carried out in rabbit reticulocyte lysates at 120 mM KCl. An arrow indicates the core-LUC fusion protein. Computer imaging was generated by Adobe Photoshop, version 3.0. (D) Relative LUC activity of the translation products of various RNAs. The LUC activity of HCV-5CL RNA is artificially set at 100%. The columns and bars represent the means and standard deviations of two sets of triplicate studies. The asterisks indicate that the translational enhancement of these RNAs compared to the translational level of HCV-5CL RNA is significant. ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 1

FIG. 2

Primer extension study of the HCV RNA constructs. (A) Calibration of the primer extension reactions. Decreasing amounts of HCV-5CL-X RNA were used in the primer extension reactions with a 5′-UTR primer, yielding a 265-nt product (arrow). (B) RNA stability of HCV-5CL (lanes 1 to 3), 5CL-Vec (lanes 4 to 6), and 5CL-X (lanes 7 to 9) RNA in rabbit reticulocyte lysates. Two micrograms of each RNA was used in in vitro translation in rabbit reticulocyte lysates. Reactions were stopped at 0 min (lanes 1, 4, and 7), 30 min (lanes 2, 5, and 8), and 90 min (lanes 3, 6, and 9). RNAs were extracted, and half of the amounts from each time points were used in primer extension experiments as in panel A.

FIG. 3

Effects of the X region on HCV translation in vivo. Linearized plasmids were transfected into Huh7 cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Relative LUC activities in the lysates were determined 24 h after transfection. The columns and bars represent the means and standard deviations of three independent transfections. ∗, P < 0.05 compared with HCV-5CL.

FIG. 4

Effects of the X region on translation from an EMCV IRES. (A) Schematic diagrams of the plasmids used. pGL-EMCV contains the T7 promoter (large open arrow), the 5′-UTR of EMCV (single line), and LUC genes (closed box) in the pGL vector. pGL-EMCV-X contains, in addition, the X region of HCV at the 3′ end. The plasmids were linearized with the appropriate restriction enzymes and transcribed with T7 RNA polymerase to generate transcripts. (B) In vitro translation products of the various RNAs were separated by SDS-PAGE on 7.5% polyacrylamide gels. In vitro translation was performed in rabbit reticulocyte lysates at 120 mM KCl. An arrow indicates the LUC protein. Computer imaging was generated by Adobe Photoshop, version 3.0. (C) Relative levels of LUC expression of the various RNAs. The activity of the EMCV transcripts is set at 100%. The columns and bars represent the means and standard deviations of two sets of triplicate studies. ∗∗, P < 0.01 compared with EMCV RNA.

FIG. 5

Effects of the X region on α-globin translation from the 5′-UTR of the α-globin gene. (A) Schematic diagrams of the plasmids used. pGL-αglobin-X contains T7 promoter (large open arrow), the 5′-UTR (single line) and coding region (open box) of α-globin gene fused to LUC genes (closed box), and the X region of HCV in the pGL vector. The plasmids were linearized with the appropriate restriction enzymes and transcribed with T7 RNA polymerase to generate uncapped and capped RNAs. (B) In vitro translation products of uncapped (left) and capped (right) RNAs separated by SDS-PAGE on 7.5% polyacrylamide gels. Translation was performed in rabbit reticulocyte lysates at 70 mM KCl. An arrow indicates the α-globin–LUC fusion protein. Computer imaging was generated by Adobe Photoshop, version 3.0. (C) Relative LUC expression of uncapped (left) and capped (right) RNAs. α-Globin RNA is set at 100%. The columns and bars represent the means and standard deviations of two sets of triplicate studies.

FIG. 6

Effects of the X region on translation from bicistronic RNAs. (A) Schematic diagrams of plasmids used. pCAT-5CL contains the T7 promoter (large open arrow), the CAT gene (hatched box), the 5′-UTR (single line), and the core protein-encoding region (open box) of HCV fused to a LUC gene (closed box) in the pGL vector. pCAT-5CL-X contains, in addition, the X region at the 3′ end. The plasmids were linearized with the appropriate restriction enzymes and transcribed with T7 RNA polymerase to generate transcripts. (B) In vitro translation products of RNAs with 50 mM KCl (left) or 120 mM KCl (right) after separation by SDS-PAGE on 10% polyacrylamide gels. The core-LUC fusion protein (upper arrow) and CAT (lower arrow) are indicated. (C) Relative LUC expression of RNAs with 50 mM KCl (left) or 120 mM KCl (right). The columns and bars represent the means and standard deviations of two sets of triplicate studies. ∗, P < 0.05; ∗∗, P < 0.01 (compared with CAT-5CL RNA).

FIG. 7

Effects of the X region on translation in vivo from bicistronic RNA constructs. Linearized DNAs were transfected into Huh7 cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Luciferase and CAT activities were determined at 24 h posttransfection. The relative LUC and CAT activities of the various RNAs and their LUC/CAT ratios are shown. The columns and bars represent the means and standard deviations of three independent transfections. ∗, P < 0.05 compared with CAT-5CL RNA.

FIG. 8

The trans effects of the X region on translation. A 1- to 10-fold excess of HCV X(+) RNA (17) was added to rabbit reticulocyte lysate containing CAT-5CL RNA (A) or CAT-5CL-X RNA (B). In vitro translation was carried out at 120 mM KCl. Translation without free HCV-X(+) RNA [(−)] is set at 100%. The columns and bars represent the means and standard deviations of three independent translation reactions.

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The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site - PubMed (original) (raw)